| BackgroundAs an important factor to maintain the stability of glucose and lipid metabolism, dietary fats are the macronutrient for human and mammals. The liver plays a key role in regulating fatty acids (FAs) metabolism. The total intake, the constituted types and ratios of the FAs in dietary fats may influence the hepatic lipid composition and also the expression of genes, which were involved in glucose and lipid metabolism. Accumulating evidence suggests that FAs overdose may have resulted in the development of metabolic diseases, such as obesity, diabetes and nonalcoholic fatty liver diseases.Although some previous studies discussed the effects of dietary FAs on glucose and lipid metabolism, the reports regarding to the prevention of lipid metabolic dysfunction and the FAs effect on the expressions of genes for hepatic glucose and lipid metabolism are limited. Therefore, the present study investigated the effects of FAs at different concentrations individually or in combinations with different ratios on the expression of genes involved in lipid metabolism. Currently, the evidence of what kinds of solvent medias for FAs and cell lines suited the study of lipid metabolism was still not clear, so we screened the cell lines and solvent medias for FAs firstly.Part1The Effects of Oleic Acid in Different Solvent Medias on the LO2and HepG2Cells Growth and the Level of Intracellular TriglycerideObjective: To find a suitable solvent media for fatty acids and an appropriate cell line in vitro for lipid metabolism research, by comparing the effects of oleic acid (OA) in different solvent medias on the human normal liver cells (LO2cell) and hepatocarcinoma cells (HepG2cell).Methods:LO2and HepG2cell lines were both divided into control group, DMSO group, OA group with DMSO, BSA group and OA group with BSA. After being cultured for24and48hours respectively, the cell viability was analyzed using MTT method, the fatty drop lets in cells were observed by oil red O staining under light microscope and the level of intracellular triglyceride (TG) was detected by reactant kits.Results:(1) Compared with the viabilities of LO2cells in control group, those treated with DMSO for24hours all increased significantly (P?O.05) and were reduced as treating lasted, especially in0.8%DMSO group (P?0.05); with increased concentration of OA with DMSO, the viabilities of LO2cells for24hours increased first, then decreased at6.4?g/ml and had no statistically significant differences compared with the control; with the treating time passing by, the viabilities were correspondingly reduced, especially at the concentration of3.2and6.4?g/ml (P?0.05). The viabilites of HepG2cells treated with DMSO for24h showed no statistically significant differences when compared with the control and were correspondingly reduced as the time passing by, especially at the concentration of0.4%and0.8%(P?0.05). The viabilities of HepG2cells treated OA with DMSO for24hours had no statistically significant differences within the concentration of3.2?g/ml and began to reduce significantly at6.4?.g/ml (P?0.05), the viability was also correspondingly reduced and significantly lower than those in the control group as the treating lasted,(P?0.05).(2) The viability of LO2cell in BSA group was increased and had no statistically significant differences compared with that in control group. With prolongation of BSA, the cell viability was significantly increased (P?0.05). The viabilities of LO2cells treated OA with BSA24h were increased with the increasing concentration of OA (P?0.05); after48h, they were correspondingly increased and significantly higher than those in control group and BSA group. The HepG2cell viability in BSA group had no significant differences compared with that in contro1group after24h and was increased significantly after48h; the viability of HepG2cells treated OA with BSA for24h was increased significantly compared with that in control group and BSA group, that was still significantly increased after48h but had significant differences only at6.4?g/ml compared with that in BSA group.(3) Oil red O staining showed the result of the number of the two cells in OA group was the same as the above. Besides, the intracellular lipid droplets of LO2and HepG2cells treated with OA with DMSO for24h were increased with increasing concentration of OA, especially LO2cells, and were correspondingly increased after48h. However, the lipid droplets of LO2cell treated for24h in the OA group with BSA at high concentration were increased and correspondingly increased obviously after48h, those of HepG2cell had no change.(4) The levels f TG in each group were similar to the trend of the oil red O staining and only LO2cell line was more sensitive.Conclusions:In our study, BSA as dissolution media is better than DMSO for fat acids, LO2cell line was more suitable than HepG2cell line in developing for in vitro model of lipid metabolism. Part2The Effects of Exogenous Fatty Acids on Cell Function and Lipid Metabolism in LO2Liver CellsObjective: In present study, we selected palmitic acid (PA, C16:0), oleic acid (OA. C18:1) and docosahexenoic acid (DHA, C22:6) as the representatives of saturated, mono-unsaturated and poly-unsaturated FAs to explore the effects of fatty acids (FAs) on the cell function and the expression of genes involved in lipid metabolism in human LO2liver cells.Methods:The concentrations of PA (OA and DHA) were0.0.4,0.8,1.6,3.2,6.4and12.8?g/mL. Then, based on the cytotoxicity of each FAs, the concentrations of mixed FAs were3.2and12.8?g/mL; three ratios of PA, OA and DHA were selected, including1:2:1,1:1:1, and1:1:2. To determine the fatty acid concentration, the viability of LO2hepatic cell was determined using MTT assay; the activities of lactate dehydrogenase (LDH). aspartate transaminase (AST) and alanine transaminase (ALT) in the media were measured by spectrophotometry; TG concentration in the supernatant was estimated using the assay kit; the expression levels of PPAR-y, LXRa,SREBP-1c, LDLR and apoC-I mRNA were determined by RT-PCR.Results:(1) At concentrations of FAs?3.2?g/ml. the treatments of single FAs increased the LO2cell viability; When the FA concentration reached3.2?g/ml, the elevated cell viabilities of PA, OA and DHA groups maintained at the same levels. At FAs concentrations>3.2?g/ml, the cell viability after OA treatment still increased, but was reduced after PA or DHA treatment:when the concentration was reached at12.8?g/ml. the viabilities of PA, OA and DHA groups (31.7±5.8,303.9±14.2,4.9±0.7) differed significantly (P<0.05) with the complete loss of the viability of DHA group. Meanwhile, the FA mixtures with three ratios (PA:OA:DHA=1:2:1,1:1:1and1:1:2, respectively) increased the cell viability compared with the control. When the concentration of the total FAs was at3.2?g/ml, the cell viabilities of the combined groups were similar to those groups containing the individual FAs. When the concentration reached12.8?.g/ml, the cell viabilities of all three combined FA groups were higher than those of PA and DHA groups but lower than those of OA group.(2) After24hours, as the concentration of OA increased, the AST and LDH level decreased. When the PA and DHA concentrations were above6.4?g/ml, the levels of LDH, AST and ALT were increased significantly. When at3.2?g/ml, the FAs combinations in different ratios decreased the AST levels and increased the LDH levels (P?0.01) compared with the individual FAs. At12.8?g/ml, the level of AST in FAs combination group was lower than that in PA and DHA groups. The ALT levels of the1:2:1and1:1:1groups at12.8ug/ml were lower than those of PA and DHA groups, but higher than those of the control group.(3) The intracellular triglyceride (TG) levels after all treatments significantly increased and the accumulation obviously increased with high doses of DHA.(4) Compared with the control, the treatment of FAs increased the expression level of PPAR-y mRNA except for the1:1:1FA combination with3.2?g/ml and the1:1:2FA combination with12.8?g/ml; the expression levels of LXRa, LDLR and apoC-I were significantly decreased in all groups except for the groups of DHA and1:2:1at12.8?g/ml; except for the other groups of FAs, DHA incresed SREBP-lc mRNA expression significantly.Conclusions:Types, concentration and mixture ratios of FAs all played important roles in the cell viability and lipid metabolism-related gene expression in LO2hepatocytes.Unlike the other fatty acid, high concentrations of DHA can stimulated intracellular lipid synthesis and the expression of genes (PPAR-y, LXRa, SREBP-1c. LDLR and apoC-1) related to lipid metabolism. The high concentration of1:1:2mixed FAs can maintain the normal expression level of genes related to lipid metabolism. Part3The Effect of Exogenous Fatty Acids on on Cell Function and Glucose and Lipid Metabolism in Primary Liver Cells of RatsObjective:The aim in this study was to explore the impact of exogenous fatty acids on the cell function and the expression of genes involved in glucose and lipid metabolism in primary liver cells of rats and to provide dietary reference for the prevention of diabetes.Methods:Liver cells of SD rats were separated by using the modified two-step collagenase perfusion method and cultured according to the pre-experiment. The treatment of primary liver cells of rats was the same as the LO2cell experiment. The cell viability was determined using MTT assay, the activities of lactate dehydrogenase (LDH), aspartate transaminase (AST) and alanine transaminase (ALT) in the media were measured by spectrophotometry, the expression levels of GCK, SREBP-1c, PPAR-? and LXR? mRNA were determined by qRT-PCR.Results:(1) The cell viabilities were all increased at low concentrations. When the concentrations of PA and OA were exceeded3.2?g/ml, the trend of cell viability was decreased, but the trend of cell viability was increased with increasing concentration of DHA (P?0.01).(2) The levels of LDH, AST and ALT in PA, OA and DHA groups were not increased. In FAs combination groups, AST and ALT levels also were not increased. LDH levels in the groups of combination FAs at3.2?g/ml were all significant higher than those in control group (P<0.05). Besides, LDH levels in the groups of combination FAs at12.8?g/ml had no significant change compared with those in control group and were higher than DHA at12.8?g/ml.(3) Except for1:1:1group, the expressions of GCK in other groups at12.8?g/ml were significantly up-regulated and higher than those in control group and corresponding group at low concentrations (P<0.05); except for the groups of1:1:1at two concentrations, DHA and1:1:2at12.8?g/ml, the levels of SREBP-lc mRNA in other groups were significantly down-regulated (P?0.05); the levels of PPAR-y mRNA in all FAs groups were significantly down-regulated (P?0.05), and the levels of LXRa mRNA were increased at12.8?g/ml in FAs groups.Conclusions:Concentration, type and proportion of fatty acids greatly influenced the GCK mRNA expression in primary liver cells of rats. The GCK mRNA in1:1:1group was the most significantly up-regulated among the groups of low concentration of FAs; under the circumstance of DHA or mixed fatty acid (1:1:1or1:2:1), the expression of GCK mRNA was rely on the expression of SREBP-1c mRNA to a certain extend; As the same result of LO2cell, the type, dose and ratios of exogenous fatty acids are able to influence the genes related to lipid metabolism in primary liver cells of rats. |
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