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The adverse effects of fatty acid accumulation in non-adipose tissues lead to cell dysfunction or cell death as known lipotoxicity, which is commonly related to chronic liver disease - called non-alcoholic fatty liver disease (NAFLD). The role of triglyceride (TG) is controversial as toxic or protective to the cells. In this study, I examined role of the accumulated-TG in fatty acid treatment of HepG2 cell line. To investigate this, HepG2 hepatoma cells were treated in the presence of media supplemented with palmitate (PA) or oleate (OL) bound to albumin according to single and sequential treatment. The relationship between TG accumulation and fatty acid inducing various toxic effects was measured by photometric and microscopic methods such as LDH, MTT, TG content, or reactive oxygen species (ROS) assays, confocal microscopy and etc. In the single treatment method, stored-TG increased approximate 4-fold in treated-oleate in comparison with treated-palmitate correlated with protection from lipotoxic by reducing cytotoxicity, no change of ROS generation and not inducing cell death. In contrast, in the sequential treatment method, role of TG accumulation was shown as a dynamic by exchange of its free fatty acid with exogenous free palmitate acid flux leading to alteration of toxicity. High proportion of saturated fatty acid (palmitate/oleate) in TG deposit increased palmitate-induced toxicity of the treated palmitate. Based on these results I conclude that nature of TG is a dynamic, and the ratio of saturated and unsaturated fatty acid of the TG might be a critical factor to effect on fatty acid caused-lipotoxic.
Lipotoxicity refers to the toxic effects of excessive lipid droplets accumulated in the cytoplasm of the cells (van Herpen et al., 2008; Schaffer et al., 2003). Two types of the possibly unrelated cellular injuries associated with these excess including apoptosis, and impaired insulin signaling (Li et al., 2010). Therefore, it is known as the hallmark of NAFLD - a disease associated with serious cardiometabolic abnormalities, including type 2 diabetes mellitus (T2DM), the metabolic syndrome, and coronary heart disease (Fabbrini et al., 2010; Malhi et al., 2008). Also, this disease consists of a wide spectrum of conditions associated with over-accumulation of lipids in the liver ranging from simple steatosis to an intermediate lesion (nonalcoholic steatohepatitis or NASH), and eventually to cirrhosis (Choi et al., 2008). Hepatic lipid accumulation can be caused by four different metabolic perturbations: adipose TG lipolysis, hepatic de novo lipogenesis; decreased hepatic fatty acid oxidation; and inadequate TG secretion in VLDL (Nagle et al., 2009).
The mechanism of lipotoxicity as well as NAFLD is still unclear. Although based on either the majority form of stored-triglycerides, or accumulation of several other lipid metabolites including different free fatty acids (FFA), diacylglycerol, free cholesterol (FC), cholesterol ester (CE), ceramide, and phospholipids, many possible mechanisms of intracellular injury were proposed (Alkhouri et al., 2009; Malhi et al., 2008). For instance, those mechanisms are comprised of increased fatty acid oxidation and oxidative stress, alteration of cellular membrane fatty acid, and phospholipid composition alteration of cellular cholesterol content, disturbances in ceramide signaling, and direct free fatty acid toxicity.
Triglyceride (TG) is a neutral lipid comprising one glycerol backbone and three long chain fatty acids (Choi et al., 2008). TG synthesis has been described including two major pathways - the glycerol phosphate and the monoacylglycerol pathway (Chen et al., 2000). In the first pathway, two fatty acyl CoA reacts with glycerol-3-phosphate to form phosphatidate that is subsequently catalyzed to form diacylglycerol through dephosphorylation. In the second pathway, dietary monoacylglycerol is acylated in enterocytes of the small intestine to produce diacylglycerol. In the final step in TG synthesis, 1,2-diacylglycerol and fatty acyl CoA are joined to form TG by catalyst of DGAT (acyl CoA:diacylglycerol acyltransferase). At least two different acyl-CoA-dependent DGATs, namely DGAT1 and DGAT2, are found in mammalian cells (Athenstaedt et al., 2006). TG lypolysis is carried out by lipases, which catalyzes TG to release free fatty acids and mediate glycerol. Many lipases were discovered in various tissues such as adipose triglyceride lipase, hormone-sensitive lipase, triacylglycerol hydrolase (TG hydrolyse), and arylacetanide deacetylase. Especially, TG hydrolase activity highly expresses in liver as well others tissues (Dolinsky et al., 2004). Both TG synthesis and lipolysis also were considered as important lipotoxic sources (Reid et al., 2008; Trauner et al., 2010).
Incorporation of exogenous different fatty acids into TG has a crucial effect on its composition (nature of TG). Gavino GR et al. (1991) reported TG accumulation in rat peri-renal and epididymal pre-adipocytes were treated with unsaturated fatty acid (oleic (C18:1) and a-linolenic (C18:3w3)) in the presence of 0.8 mM insulin. Cellular TG accumulated-the fatty acids were with relative enrichments over control from 1.4-fold for C18:1 to greater than 40-fold for C18:3w3. The incorporation into cellular TG correlated with increase of the concentration of treatment fatty acid. Similarly, in 3T3-L1 cells were incubated with [1-13/14C] acetate, myristate (C14:0), palmitate (C16:0), stearate (C18:0), or oleate (C18:1). Incubating cells with the long chain fatty acids resulted in its accumulation to the level of about 50% of the cellular TG fatty acids as compared to the TG stored-fatty acids in 5.0 mM treated-acetate. Saturated fatty acids at the glyceride sn-1,3 position and unsaturated fatty acids at the sn-2 position was preferentially esterified by cellular enzymes, respectively (Soma et al., 1992). Moreover, when 1.0 mM oleate and 1.0 mM linoleate was co-provided at the same time, TG-incorporated [1-13C] oleate decreased in comparison with alone oleate treatment as well the ratio of [1-13C] oleate esterified at the sn-1,3 position increased because of competition between linoleate and oleate for esterification (Guo et al., 1999).
Fatty acid composition of TG can be identified by many methods. The simplest method was used that quantifies total "double bond" fatty acids in serum lipid based on "sulfo-phospho-vanillin" reaction (Knight et al., 1972). This method was recommended that it is simple, rapid and reasonably precise. Besides, other methods were used commonly to analyze fatty acids including gas chromatography (GC), liquid chromatography (LC) and gas chromatography-mass spectrometry (GC-MS) (Arab et al., 2002). These methods require fatty acid standards having appropriate structure to fatty acids in samples. GC-MS is a powerful tool to correctly identify specific fatty acids or to explain the chemical structure of fatty acid metabolites.
Different TG particles are known as phenomenon of intracellular disorders. In human study, NAFLD progression was determined association with numerous changes in hepatic lipids of liver, while increase of saturated fatty in TG was driven by palmitate (Puri et al., 2007). In vivo, Larter et al. (2008) demonstrated hepatic acid composition was altered by dietary lipid source in obesity mice, increasing ratio of monounsaturated:saturated free fatty acid reduced nuclear levels of the lipogenic transcription factor sterol regulatory element binding protein-1 (Larter et al., 2008). Recently, in vitro research, Ricchi et al (2009) suggested with a given amount of intracellular TG, the degree of apoptosis only depended on fatty acid structure, but not TG content.
Yet, the role of TG accumulation in this disease is still controversy about whether toxic or protection. In clinical study, TG deposit is characterized as a toxic species based on two-hit hyphothesis (Day et al., 1998). According to the hypothesis, in the early stage, an excess of TG is synthesized in hepatocytes, so the mechanism promoted TG removal (oxidation or secretion) does not balance with promoting lipid import. Hence, steatosis is generally considered to be a rick factor for more serious form of liver damage. Furthermore, TG deposit also associates with insulin resistance. The decreasing sensitivity to insulin in adipocyte parallels with increasing stored-TG within adipose depots (Samuel et al., 2004). Development of insulin resistance increased risk of developing NAFLD from a 4- fold to an 11-fold (Nagle et al., 2009). In vivo experiment demonstrated that inhibiting TG synthesis by blocking DGAT2 in MCD diet-fed mice improves hepatic stetosis by reducing hepatic expression of tumor necrosis factor alpha, increasing serum adiponectin, and striking improvement in systemic insulin sensitivity (Yamaguchi et al., 2007). On contrary, in vitro study, TG accumulation rescued cell death from lipotoxic in varied cell types (Cnop et al., 2001; Gómez-Lechón et al., 2007; Listenberger et al., 2003). TG synthesis directly decreases excess fatty acid incorporation into some mediate molecules such as diacylglycerol (DAG) and shingolipids (Holland et al., 2008; Petschnigg et al., 2009). Additionally, stored-TG influenced on the direction of free fatty acid flux as well promoted fatty acids towards beta-oxidation at mitochondria in order that block fatty acid induced-toxic (Henique et al., 2010).
However, those distinctions between in vivo and in vitro have been accepted for long time without explanation. Therefore, a consideration for in vitro model is necessary. Most methods for treating free fatty acid in accumulated-TG research were used as following single or co-treatment in in vitro (Cnop et al., 2001; Gómez-Lechón et al., 2007). Within those methods, lipotoxic from free fatty acids or stored-TG is not able to distinguish, since intracellular trafficking of free fatty acids after uptake has multi-target, and complex (McArthur et al., 1999). For example, treated-oleate was non-toxic due to having a large amount of TG accumulation (Gómez-Lechón et al., 2007; Listenberger et al., 2003). However, oleate can regulate specific gene expression - the fatty acid binding protein adipocyte P2 (aP2) plays a role in fatty acid transport or protection against the detergent-like effects of fatty acids (Distel et al, 1992; Matarese et al., 1989). Thus, it is complicate to distinguish result of the protection from whether TG deposit or gene expression. In this study, I applied a new treatment system-sequential treatment to test the role of TG in in vitro. This model was described in a previous study about lipotoxicity mechanism (Henique et al., 2010).
In the present study, firstly, I investigated whether TG accumulation essentially plays a protective role in single treatment of free fatty acid in HepG2 cell line. Secondly, sequential treatment was employed to discover whether fatty acid exchange of TG with exogenous free fatty acid occurs. Finally, I examined whether different percentage of fatty acid in nature of TG changes lipotoxic.
Materials and methods
HepG2 cell line was purchased from American Type Culture Collection (ACTT) (VA, USA). Some chemicals were obtained from Invitrogen Co. (Oregon, USA) including Dulbecco's Modified Eagle Medium (DMEM), 0.5% Trypsin-EDTA 10x, Penicillin-Streptomycin (PS), BODIPY@493/503, and Carboxyl-H2DCFDA. The listed-chemicals came from SIGMA-ALDRICH, Inc., (MO, USA) as following: Fatty acids (Palmitic, Oleic acid), Dimethyl sulfoxide (DMSO), Propidium iodide (PI), Paraoxon-ethyl (E600), and Triton X-100. Bovine serum albumin, fraction V, fatty acid free (BSA) and Cytotoxicity detection kit were from Roche (IN, USA). Fetal Bovine Serum (FBS) was gotten from Welgene Inc. (Daegu, South Korea). MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay) was purchased from Molecular Probes (Oregon, USA). Triglyceride Quantification Kit was purchased from BioVision Inc. (CA, USA). Phosphate buffered saline (PBS) was made up of chemicals at pH 7.4, including 8.0 g NaCl, 0.2 g KCl, 1.44 g Na2HPO4, and 0.24 g KH2PO4 in 1 liter deionized water. All other chemicals met in standard grade of analysis.
Culture of HepG2 cells
HepG2 cells were cultured in DMEM containing 10% (v/v) FBS and 1% (v/v) Penicillin-Streptomycin under 5 % CO2, 95 % humidity at 37Â°C.
Fatty acid treatment
The stock solutions of fatty acids were prepared at 100 mM in DMSO and stored at -20 oC. They were diluted with DMEM media at proper concentrations, and the solutions were sonicated for 1 hr. After that, the diluted solutions were mixed with BSA at 2:1 ratio of fatty acid to BSA in all experiments. Finally, they were constantly shaken for 1 hr on a shaker before adding into the cell.
When 80% confluency was reached, the cell was overloaded with free fatty acids in two treatment systems. In single treatment, the cell was treated with a unique fatty acid in various concentrations (0 to 1.0 mM) for 24 hrs of incubation time. While in sequential treatment, HepG2 was firstly pre-incubated for 24 hrs with 1.0 mM of oleic acid, then variable concentrations of palmitic acid was supplied for 24 hrs.
Cytotoxicity was based on measurement of lactate dehydrogenase activity using a cytotoxicity detection kit (Roche, IN, USA). To conduct the assay, HepG2 was seeded at 3x104 cells/well in 96 well-plate, after desire exposure time, the culture supernatant was collected. The reaction mixture from the kit was then applied in the samples. The absorption of the formazan dye formed was measured at 490 nm on ELISA reader (VersaMax Microplate Reader, Molecular Divices., CA, USA). The percentage cytotoxicity was determined by dividing the value of the average absorbance values of samples by the high controls after the average absorbance values in the background control were subtracted from these values.
Cell viability was measured based on measurement absorption of a water-insoluble purple formazan which was reduced from a yellow water-soluble tetrazolium salt by oxido-reductases in the live cells. This experiment was modified as following protocol in a previous journal (Min et al., 2008). Briefly, the cells were seeded at 3x104 cells/well in 96 well-plate. After 24 hrs, they were treated with MTT stock (5 mg/ml) to form furmazan crystals in DMEM at 37 oC for 1.5 hrs. Next, DMSO was added to dissolve the crystals. The absorbency was measured at 570 nm using ELISA reader. The estimation of cell viability was calculated by comparing between the spectra value of treated and untreated cells.
Quantification of triglyceride
Intracellular triglyceride content was determined according to an enzymic method (BioVision Inc., CA, USA). In this method, enzyme lipase catalyzes TG to form glycerol which reacts with the probe to generate coloration measured on spectrophotometry at 570 nm. In briefly, the cells were harvested, and homogenized in 5% Triton X-100 solution. After slowly heating several times at 80-100Â°C, the supernatant was collected from removing insoluble materials by centrifuging at 12000 rpm. Finally, the coloration of sample was formed by mixing reaction mixture with lysed-supernatant by lipase. The value of TG content was quantified based on TG standard curve that was constructed with different concentrations of TG (0, 0.02, 0.04, 0.06, 0.08, and 0.1 mmol/ml).
Measurement of reactive oxygen species (ROS) generation
The measurement of ROS production within cells was carried out by using 2â€²,7â€²-Dichlorohydrofluorescein diacetate (Carboxyl-H2DCFDA; Invitrogen Corporation, Oregon, USA) which is combined into fluorescent products in the presence of H2O2 and other ROS molecules and esterases (Song et al., 2007). We followed a procedure in literature with a minor modification (Song et al., 2007). Briefly, the cells were overloaded with various concentrations of fatty acids, after that, 100 mM final concentration of Carboxyl-H2DCFDA was added in the media without fetal bovine serum at 37 0C in darkness for 30 min. Then, the cells were washed twice with warmed PBS and lysed in RIPA buffer (PIERCE, IL, USA). The lysed-cells were centrifuged at 12000 rpm for 5 min. Finally, the supernatants were conveyed to a 96-well back plate, and excited at 485 nm and emitted at 530 nm for the Carboxyl-H2DCFDA fluorescence on Fluorometer (VICTOR2, Perkin Elmer., MA, USA).
Intracellular triglyceride staining
BODIPY@493/503 (Invitrogen Co., Oregon, USA) was used to capture TG fluorescence on Confocal microscopy. In this experiment, the cells were prepared as above. Before the dyes treatment, the cells were washed with PBS twice times. Bodipy @493/503 was then added at 1.0 mM final concentration and 10 min of incubation at 37 oC after that the cells were rinsed with PBS again. Zeiss LSM Image Brown software (LSM 510 Meta, Carl Zeiss Inc., Jena, Germany) was handled to take TG image at excitation of 488 nm and emission of BP 505-530 nm.
Confocal microscopy of reactive oxygen species
ROS generation in HepG2 cells was demonstrated using intracellular oxidation of carboxyl-H2DCFDA (Hyun et al., 2010). Briefly, the cells were washed twice with warmed PBS. Then, the cells were treated with carboxyl-H2DCFDA at 10 mM final concentration in serum free DMEM media and incubated for 30 min at 37 oC under dark condition. Next, the excess dye was rinsed with warmed PBS and finally, the fluorescence images of the cells were taken using confocal microscopy. The samples were excited at 488 nm and the emission filter was LP 530 nm. 100 mM hydrogen peroxide treatment for 1 hr in the same condition was adopted as the positive control.
Detection of triglyceride accumulation and cell death by confocal microscopy
TG droplets and cell death were detected on confocal microscopy by using co-staining BODIPY@493/503 (Invitrogen, Oregon, USA) and propidium iodide (PI), respectively. Firstly, HepG2 seeded at 1x105 cells/well in the 24-well plate, and treated with final concentration of fatty acids to 1.0 mM for 24 hrs in the treatment systems. Secondly, the cells were stained with 1.0 mg/ml final concentration of BODIPY@493/503 dissolved in PBS, after washed several times with PBS. This process was kept in darkness for 10 min at room temperature. Subsequently, the bodipy solution was removed, and the cells were then washed by PBS. Finally, the cells were incubated in 40 mg/ml of PI for 10 min in darkness. Exposition of TG accumulation and cell death was observed at excitation of 488 and 543 nm, and emission of BP 505-530 and LP 650 nm on the confocal microscopy, respectively.
Inhibitor of TG hydrolase treatment
Paraoxon-ethyl (E600) was used to inhibit TG hydrolase according to a previous study (Gilham et al., 2003). The protocol was modified as descriptions below. Concisely, the cells were seeded on 96-well plate, and treated fatty acids as following the sequential treatment. Before adding fatty acid in the last period, the cells were incubated with different concentrations of E600 in DMEM for 1 hr. Consequently, the fatty acid solutions containing various doses of inhibitor were supplied to replace the old media. Ultimately, the cells were kept for 24 hrs incubation.
All results were expressed as mean values Â± standard error (n=4 or 5). The difference between the groups was evaluated using Student's t-test.
Toxicity of various fatty acids
To examine influence of dose dependence and time course on fatty acid caused-toxicity, I conducted two experiments in HepG2 including cytotoxicity (Figure 1-1) and ROS generation (Figure 1-2).
In figure 1-1, the cytotoxicity of the various free fatty acids (palmitate and oleate) at different concentrations (0, 0.1, 0.2, 0.3, 0.5, 0.7 and 1.0 mM) was also investigated after 24 hrs and 48 hrs of treatment. The results show that the various doses of the FFA produced different cytotoxic and cell viability effects. As the figure presents, the toxicity of palmitate significantly increased above 0.5 mM, and at 1.0 mM palmitate was shown to be highly toxic as well 15% increase in comparison with control samples. In addition, the toxicity in the second day treatment was twice as high as in the first day (48.70Â±2.17%). In contrast, cytotoxic effect of oleate was virtually no change at the concentration from 0 up to 1.0 mM. Similarly, only palmitate was found to significantly decrease cell viability by above 35% and nearby 65% at 1.0 mM for the first and second day, respectively, although the slight decline also was observed in oleate for both days. Briefly, no cytotoxic effect was observed in HepG2 at the concentration of oleate from 0 up to 1.0 mM, while a strong cytotoxic effect was shown at 1.0 mM of the palmitate for two days. Based on these results, 1.0 mM palmitate for 24 hrs incubation was selected for further study. Moreover, my data indicated cell viability was not sensitive to quantify cytotoxicity in lipotoxic.
The range of concentrations of fatty acid generated-ROS was explored after 24 hrs on Figure 1-2. As the results show, fluorescence exposure of ROS on confocal microscopy was much more in 1.0 mM treated-palmitate, but not in oleate in comparison with positive control (Figure 1-2A). In the same way, fluorescent intensity displays, the dose dependence of fatty acid generated ROS was only effective in palmitate (Figure 1-2B), while the concentration 0.5 mM up to 1.0 mM was found a significant increase by over 1.5-fold to around 3-fold, respectively. This result was similar to the effect of fatty acids on cytotocixic showed in Figure 1-1. These findings imply that palmitate generated-ROS completely associates with cytotoxicity increase.
Figure -1. Cytotoxicity of free fatty acids at different concentrations. The cells were seeded at 3 x 104 cells/well on a 96-well plate and incubated under 5% CO2, 95% humidity at 37Â°C. When 80% confluency was reached, they were treated with different concentrations of free fatty acids (0, 0.1, 0.2, 0.3, 0.5, 0.7, and 1.0 mM) dissolved in DMEM media containing a constant ratio of fatty acid bound BSA at 2 to 1. After the incubation time, cytotoxicity was assessed by MTT and LDH assay. A, B and C, D - cell viability and cytotoxicity in palmitate and oleate treatment for 24 hrs and 48 hrs, respectively. Values are means Â± SE. p - Student's t-test. *p< 0.05 vs. control; #p< 0.01 vs. control.
Figure 1-. Reactive oxygen specie (ROS) generation by free fatty acids. The cells were treated with 1.0 mM of free fatty acids for 24 hrs. Before observing on confocal microscopy, added 10 mM of Carboxyl-H2DCFDA dye was incubated for 30 min to capture ROS fluorescence. ROS positive control samples were prepared in serum free media containing 100 mM of hydrogen peroxide for 1 hr incubation. ROS florescence (A) was showed by Carboxyl-H2DCFDA excited at 448 nm, and selected filter emission at LP 530 nm. The images showed fluorescence and phase contrast on confocal microscopic software. (a) H2O2 treated samples, (b) Control cells, (c) 1.0 mM treated palmitate and (d) 1.0 mM treated oleate; I and II - ROS fluorescence channel and merged fluorescence channel, respectively; Original magnification 20X. The quantitative of ROS fluorescence of palmitate (B) and oleated (C) at various concentrations. Arbitrary unit (a.u) are means Â± SE. p - Student's t-test. *p< 0.05 vs. control, **p< 0.01 vs. control, ***p< 0.005 vs. control.
Protection of TG deposit from saturated fatty acid induced lipotoxic
In order to study relationship of accumulated-TG and cell death, I measured intracellular TG content, and exposed stored-TG and cell death on confocal microscopy after 24 hrs fatty acid treatment. In Figure 2A shows, TG was accumulated much more in oleate, but less than in palmitate in comparison with control cells. Particularly, the mount of TG content in oleate was nearly 4.5-fold in palmitate, and 17-fold in control as high as in the order of comparison. Moreover, by co-staining bodipy and PI to detect TG droplet and cell death, fluorescence image showed relationship between TG and cell death. As Figure 2B presents, the fluorescence of PI staining (red color) was only exposed in palmitate, and did not overlap with bodipy fluorescence (green color). In other words, palmiate induced-cell death did not parallel with TG deposit. These findings indicate that TG accumulation plays a protective role in lipotoxicity.
Figure 2. Intracellular triglyceride and cell death during 24 hrs incubation with free fatty acids. The cells were treated with 1.0 mM of free fatty acids for 24 hrs. A - Fluorescence exposure of TG on confocal microscopy; the measurement of TG content was determined by using enzyme method as the description in material and method section. Values are means Â± SE. p - Student's t-test. ***p< 0.005 vs. control. B - Fluorescence exposure of TG (green color) and cell death (red color) co-staining in 1.0 mM palmitate treated cell. After the incubation time, 1.0 mg/ml of bodipy and 40 mg/ml of PI were in that order added to stain TG and cell death. The cells were continued to incubate in the conditions protected from the light until fluorescence intensity showed maximum on microscopy. Before the cells were observed on confocal microscopy, the mixed dyes solution was replaced with 1X PBS buffer. Bodipy was excited at 488 nm and a BP 505-530 nm emission filter was used, whereas PI fluorescence was exposed at LP 650 nm emission and 543 nm excitation. The images showed fluorescence and phase contrast on confocal microscopic software. I - Bodipy fluorescence channel, II - PI fluorescence channel and III - phase contrast; Original magnification 20X.
Palmitate-induced toxicity under pre-accumulated TG
Role of TG still is controversy about toxic, inert, or dynamic in lipotoxicity (Day et al., 1998; Neuschwander-Tetri, 2010; Reid et al., 2008). To discover whether TG droplet plays the functions as above mention, I applied new system treatment - named sequential treatment, in which free TG accumulation was established inside cells by 1.0 mM oleate of supplementation for 24 hrs. After that, the oleate solution was replaced with 1.0 mM of palmitate, and the cells were remained in the new solution for next one day of incubation. The explanation for this system was described in diagram below (Figure 3A). In these experiments, I measured the LDH release into the medium after the exposing time of various doses of palmitate (0, 0.1, 0.2, 0.3, 0.5, 0.7 and 1.0 mM). TG content and its relation with cell death were examined at 1.0 mM fatty acid sequential treatment as well. As result shows in Figure 3B, interestingly, the dose dependence of palmitate caused-cytotoxicity dramatically increased from 0.7 mM to 1.0 mM by around 5% to above 12%, respectively. This finding was dissimilar to the previous results about the protective TG. Otherwise, the highest toxic at 1.0 mM of palmiate was approximately decrease by 5% than its value in alone treated-palmitate (Figure 1-1). Based on this result, I suggest that pre-accumulated TG might have implication in reducing palmitate-caused toxicity. Furthermore, according to quantity of TG content showed in Figure 3C, about 1.5-fold of the proportion of TG was created from sequential treatment of 1.0 mM oleate and palmitate in comparison with oleate alone. This result implies that amount of free palmitate was converted to TG deposit, even the excess of TG amount still had presented inside cells. In addition, as fluorescence exposure of TG-cell death in Figure 3D shows, a large of cell number was estimated to display the overlap between TG (green color) and cell death (red color). While, the dominant number expressed on mild death cells that the stained-DNA was not high condensed. Collectively, this evidence reveals that free TG had an active participation in reducing palmitate caused-cell death, although did not completely success in preventing cell death. To sum up, these findings suggest that free TG had an effect on lipotoxic.
Figure 3. Free fatty acid induced-toxicity in sequential treatment. The cells were treated with 1.0 mM of oleic acid for 24 hrs. After the incubation time, the old medium was replaced with the various concentrations of palmitate dissolved in DMEM media containing the constant ratio of fatty acid bound BSA for 24 hrs. A - Diagram of sequential treatment. B and C - Cytotoxicity in dependence dose of palmitate and measurement of triglyceride content in 1.0 mM fatty acid treated cell, respectively. Values are means Â± SE. p - Student's t-test. ###p< 0.005 vs. control; *p< 0.05 vs. 1.0 mM treated oleate; ***p< 0.005 vs. 1.0 mM treated oleate. D - Fluorescence exposure of triglyceride (green color) and cell death (red color) co-staining in 1.0 mM sequential treated oleate and palmitate, respectively. I - Bodipy fluorescence channel, II - PI fluorescence channel, and III - phase contrast; Original magnification 20X.
Dynamics of free TG accumulation results in changing toxic of fatty acids
As the previous results showed that free TG involved in lipotoxic decrease (Figure 3B and 3D), but this was not entirely true since free palmitate was promoted toward TG synthesis. I hypothesized that free TG exchanges its unsaturated fatty acid with free palmitate leading to diminish lipotoxic due to increasing high concentration of intracellular unsaturated fatty acid. To test this hypothesis, I conducted to inhibit TG hydrolase by using different concentrations of its inhibitor (E600) on fatty acid sequential treatment. As result in figure 4B shows, cytotoxicity increased by over 7% to around 16% correlated with the rise of treated-E600 dose from 10 to 40 mM in comparison with no inhibitor treatment. Specifically, no toxicity was found at those concentrations in normal cells (Figure 4A). This demonstrated that the inhibitor was effective in inhibiting TG hydrolase. Thus, a large amount of free unsaturated fatty acid released from free TG was completely blocked so that palmitate caused-toxicity intensively increased. These findings imply that free TG directly implicates in fatty acid exchange with free palmitate. Furthermore, in order to examine whether fatty acid composition of nature TG influences on lipotoxic regulation, cytotoxicity of different ratios of 1.0 mM fatty acid mixture formed-TG was investigated in present or not E600 at 25 mM. As in figure 4C presents, variable cytotoxic was observed on different proportions of fatty acid synthezed-TG. A high percentage of saturated fatty acid increased cytotoxicity without using the inhibitor, approximate 1.5-fold and 2.0-fold cytotoxicity increase was observed in 1.0 mM fatty acid mixture (0.5 mM OL together with 0.5 mM PA) and (0.25mM OL together with 0.75 mM PA) as compared to the mixture (0.75 mM OL together with 0.25 PA), respectively. Likewise, cytotoxic increase was also showed in samples treated with E600. The increase response to the mixture (0.5 mM OL together with 0.5 mM PA) was 4% high as compared to the mixture (0.75 mM OL together with 0.25 PA), while (0.25 mM OL together with 0.75 PA) increase an 8 of percentage. Unexpectedly, cytotoxicity in the percentage of unsaturated fatty acid at 100 in the mixture was an exception of the trend. Collectively, these data suggest that various proportions of fatty acid mixture synthesized-free TG extremely impacts on lipotoxic regulation.
Figure 4. Effect of free TG accumulation on fatty acid caused-cytotoxic. The cells were seeded, and treated fatty acids according to the sequential treatment. A - Cytotoxicity of different concentrations of E600 in normal cells for 24 hrs. B - Dose dependence of E600 influences on 1.0 mM palmitate induced toxicity. C and D - Toxicity of various ratios of fatty acid mixture without and in present treated-E600 at 25 mM, respectively. Values are means Â± SE. p - Student's t-test. #,*p< 0.05; **p< 0.01; ***,+++p< 0.005; *p vs. control; + vs. 1.0 mM of oleate/palmitate in present or without E600 treatment; # vs. 1.0 mM of oleate/palmitate at 25 mM of E600 treatment.
The role of TG accumulation in varied cell types was known as a protection in vitro model in previous dominant literatures in lipotoxicity (Gómez-Lechón et al., 2007; Listenberger et al., 2003). Surprisingly, in this present research, my findings discovered that the different functions of TG were in the distinct system treatments. Firstly, TG plays a protective role from lipotoxic in single treatment. As the results showed, the high proportion of TG deposit was 4.5-fold as much as in 1.0 mM oleate compared with 1.0 mM palmitate (Figure 2A) was not toxicity, whereas the less accumulated-TG in palmitate significantly increased cytotoxicity (Figure 1-1C). Another evidence presented the visibility of intracellular TG on confocal dissociated with cell death (Figure 2B). Secondly, dynamics of free stored-TG was found in sequential treatment. The results in Figure 3B demonstrated that free TG actively participated in reducing palmitate caused-lipotoxic. Particularly, toxic effect of dynamic free TG was closely impacted by its unsaturated fatty acid composition, as the observation in Figure 4D. To sum up, TG accumulation not only has a protective role as many mentioned-papers, but also is a dynamic involving in fatty acid exchange, that is virtually opposite to the opinions suggesting TG seem to be inert (Liu et al., 2010; Neuschwander-Tetri, 2010).
TG protection in lipotoxicity was firstly described in a research on beta-cell nearly a decade ago (Cnop et al., 2001). Subsequently, many researches were openly conducted in various cells, and obtained the same conclusion (Gómez-Lechón et al., 2007; Listenberger et al., 2003). In my finding also agree with previous researches concluded that TG accumulation prevents cell death from lipotoxic in HepG2. Although, this is the first report to directly present the relationship between TG and cell death by using the fluorescence detection method on confocal microscopy (Figure 2B). The advantage of this technique permits to expose the visibility of intracellular TG and cell death signals in the same cells, this is never reported in prior studies. The strong evidence was obtained by using the method to restate that TG deposit is equivalent to the mechanism of protection from lipotoxic.
Up till now, the protective mechanism of TG has been also illustrated in some dissimilar ways. Firstly, TG synthesis was reported that decreases excess fatty acid incorporation into some mediate molecules such as diacylglycerol and shingolipids, which may regulate essential signaling and structural function (Holland et al., 2008; Petschnigg et al., 2009). Another way showed that pre-accumulated TG effected on the direction of free fatty acid flux as well promoted fatty acids towards beta-oxidation at mitochondria in order that block fatty acid induced-toxic (Henique et al., 2010). However, the excess amount of TG is considered as related to intracellular disorders (Choi et al., 2009).
One of serious diseases is characterized by uncontrolled accumulated-TG named NAFLD, but whose mechanism has not been clearly understood (Browning et al., 2004). Nevertheless, two-hit hypothesis are widely accepted to explain mechanism of this disease (McArthur et al., 1999). According to the hypothesis stated, TG is synthesized from lipids so much in hepatocytes in early stage of the disease. As in the result, the mechanism promoted TG removal (oxidation or secretion) does not balance with promoting lipid import. Hence, steatosis is generally considered to be a rick factor for more serious form of liver damage.
Yet, a difference of opinion argued that TG seems to be inert as same as two hit hypothesis is not still true (Liu et al., 2010; Neuschwander-Tetri, 2010). Recently, on contrary, by discovering lipase and its role in very-low-density lipoprotein secretion in hepatocytes demonstrated the dynamic of free TG - supplies an essential material (free fatty acids) to form cholesterol core in VLDL secretion (Gilham et al., 2003; Wei et al., 2007). This partly illustrated the complex function of free TG implicating in physiology as well in intracellular disorder as previous mentioned-proposals (Athenstaedt et al., 2006). In fact, some questions remain whether both fatty acids removed from free TG and composition of stored-TG can have an impact on lipotoxic.
By applying sequential treatment (Figure 3A), my study confirmed the dynamic of free TG in lipotoxicity in liver, and also found that lipotoxic of dynamic TG is influenced by its unsaturated/saturated fatty acid ratio. In my experiments presented here show that palmitate induced toxicity significantly increased by blocking TG hydrolase in comparison with the samples not having co-treated TG hydrolase inhibitor (Figure 4B). This can be explained by free fatty acid exchange between free TG and intracellular fatty acid flux. According to our experiment model, in the first period, a large amount of TG was accumulated inside liver cell by overloading oleate at 1.0 mM, the cells still were healthy under this condition (Gómez-Lechón et al., 2007; Ricchi et al., 2009). The composition of TG was determined as the dominant proportion of unsaturated fatty acid due to overloading by oleate (Guo et al., 1999). In normal condition, TG hydrolase activity highly expresses in liver that hydrolyze free TG to release high content of free unsaturated fatty (Lehner et al., 1997). In the second period, 1.0 mM supplementation of free palmitate integrated with the released-free unsaturated fatty acid leading to reduce of palmitate content in fatty mixture. The high percentage of free unsaturated acid in the fatty mixture is known as less toxic (Gómez-Lechón et al., 2007). In the similar way, by inhibiting the enzyme, toxic effect of palmitate was completely independent from released-free oleate so that palmitate induced highest toxic effect on cells.
In fact, there were several researches attempting to study TG dynamic as following different ways. Firstly, the mobilization of adipocyte TG was decided by TG hydrolase, attenuation of this enzyme activity resulted in lowering fatty acid efflux as well decrease of circulating fatty acid level (Wei et al., 2007). Secondly, TG in lipid droplets was responsible for released-fatty acids by autophagy mechanism in hepatocytes and mouse liver (Singh et al., 2009). However, these reports did not mention about the interaction between free fatty acids removing from TG and exogenous fatty acid supplement. Besides, a few of recent opinions considered stored-TG in liver as a source of lipotoxic by elevating intracellular above toxic fatty acid level, but no data have been published (Trauner et al., 2010). Collectively, our finding is the full report to indicate fatty acid exchange of TG in lipotoxicity in liver cell.
Furthermore, in this present study explored that lipotoxic of exchange between free TG and intracellular fatty acid flux is absolutely impacted by components of fatty acid in TG (nature TG). The evidence was that at high ratio of saturated\unsaturated fatty acid formed-free TG induces high toxicity by present of 1.0 mM palmitate (Figure 4C). As Gómez-Lechón et al (2007) mentioned that free fatty acid mixture containing a high proportion of saturated fatty acid (palmitate) is associated with high toxic. Also, difference profile of fatty acid in TG was examined to show that palmitate incubation induced an increase C16:1, C18:1c11 and C14:0, the incubation of OL increase C16:0, C18:0 and C14:0, while the incubation with fatty acid mixture (high proportion of oleate and palmitate) reduced the concentration of C16:1 (Ricchi et al., 2009). Therefore, our finding is appropriate with those researches to conclude much more toxic in high ratios of saturated fatty acid.
By applying sequential treatment in in vitro in lipotoxicity, our model experiment shows some advantages in simulating the interaction between free TG and lipotoxic in liver cell. This model was described in a previous study about lipotoxicity mechanism (Henique et al., 2010). Nonetheless, the selection of TG hydrolase for hydrolyzing TG is a disadvantage due to its insufficient activity for intracellular triacylglycerol lipolysis in HepG2 cell line (Gibbons et al., 1994).
In summary, due to a limitation of using sequential treatment to study TG role in lipotoxicity, no full evidence is reported TG dynamic. My study showing that TG is dynamic by actively participating in fatty acid exchange with intracellular fatty acid flux so that fatty acid induced-lipotoxic is totally interaction between supplementation of exo-fatty acids and endo-fatty acid released from TG. Moreover, fatty acid ratio of TG is a critical factor to influence on lipotoxic. The evidence shows that high percentage of saturated fatty acid in TG tends toward increase toxicity. On the other hand, an agreement on my study is that the protective role of TG in single treatment at which numerous accumulated-TG can prevents cell death from toxic fatty acids. In present research indicate an important part in complex function of TG, though it is necessary to further study.