OBJECTIVETo establish an in vitro cell model to investigate hepatic steatosis of non-alcoholic fatty liver disease.

METHODSHepG2 cells cultured in MEM containing 10 % fetal bovine serum were divided into control group and model group. At 7 0%-80 % confluency, HepG2 cells in model group were exposed to a long-chain mixture of free fatty acids (oleate and palmitate) for 24 h, cells in control group were subject to fresh medium. Lipid droplets were observed with oil red O stain and electron microscope, triglyceride and malonaldehyde were detected by respective assay kits.

RESULTA large number of lipid droplet were detected in model HepG2 cells; the level of triglyceride increased. However,malonaldehyde did not increase significantly compared with control group.

CONCLUSIONA large number of lipid droplet were detected in model HepG2 cells; the level of triglyceride increased. However, malonaldehyde did not increase significantly compared with control group.

Culture Media ; pharmacology ; Fatty Acids, Nonesterified ; pharmacology ; Fatty Liver ; Hep G2 Cells ; Humans ; Models, Biological ; Non-alcoholic Fatty Liver Disease ; Oleic Acid ; pharmacology ; Palmitates ; pharmacology

Investigation in our laboratory has been undertaken to study the effect of ethanol on the triglyceride (TG) content in the liver, the free fatty acid (FFA) content in the serum and the glycogen in the liver of rats which were fed on various diets. Four hours after administration of a sing1e dose of glucose (5g/kg BW.) and ethanol (6g/kg BW.) by gavage tube to rats fed a norma1 diet for 20 days then fasted for 18 hours, TG content in the liver increased by 80%, 10% compared to the control. When a sing1e dose of equal amounts of both glucose and ethanol were administered to another group, TG content in the liver was 42% higher than the control. There was no great change in serum FFA content in the glucose treated group as compared with the control, however, there was an increment of serum FFA content in the ethanol treated group and in the group treated with both ethanol and glucose by 81% and 71% of the control, respectively. The results indicate that ethanol administration had an inhibitory effect on the TG accumulation in the liver of rats fed by glucose. There is a correlation between TG accumulation in the liver and FFA content in the serum, and it appears that the ethanol administration did not induce the TG accumulation in the liver but the increment of serum FFA content in rats is probably due to the increased fatty acid mobilization from adipose tissue. However, countercurrent results were observed in the glucose treated group as compared with the ethanol treated group suggesting that glucose administration does induce TG accumulation in the liver but does not increase the serum FFA content in rats. The increment of serum FFA content in rats. The increment of serum FFA content by ethanol treatment was not ameliorated by glucose administration. In the liver perfusion experiment with rats fed both ethanol and various other diets, the results of incorporation of ethanol-1-14C into the total lipid in the high carbohydrate, high fat, low carbohydrate and control diet group were 1925 +/- 257 (cpm/g liver), 1237 +/- 76, 1269 +/- 105, 2041 +/- 74, respective1y. The results indicate that amount of dietary carbohydrate and high fat had an effect on the total lipid accumulation derived from ethanol-1-14C molecule in the liver. Liver glycogen content in the control on rats, high fat, 1ow carbohydrate and high carbohydrate diets were 91.5 +/- 7.9(mg%), 93.0 +/- 1.8, 99.1 +/- 4.4, and 153.7 +/- 26.0, respectively. There were no great differences between each dietary group and the rest control group except in the case of the high carbohydrate group which was over 1.5 times greater than that of the control. The incorporation of labelled ethanol into liver glycogen in the control rats and those on high fatdiet, low carbohydrate diet and high carbohydrate diet were 525, 401, 351 and 806 cpm/g liver, respectively. The increased incorporation of ethanol-1-14C into liver glycogen in the high carbohydrate diet group is thought to be due to the increased gluconeogenesis from acetyl CoA derived from 14C from ethanol because rats were fasted for 18 hours before perfusion. It might be the result of increased gluconegenesis of acetyl CoA derived from ethanol-1-14C by spare action of high carbohydrate on acetyl CoA. During the liver perfusion, 14CO2 production from ethanol-1-14C was higher in the high fat diet and low carbohydrate diet groups than in the control group, however, no great difference was observed between the high carbohydrate and control groups. The higher production of 14CO2 from the single ethanol-1-14C dose in rats on the high fat diet and low carbohydrate diet groups than in the control group is probably due to the increased metabolism of ethanol through Kreb's cycle rather than the incorporation of it into the liver fat.
Animal ; Diet ; Ethanol/metabolism ; Ethanol/pharmacology* ; Fatty Acids, Nonesterified/blood ; Glucose/pharmacology ; In Vitro ; Liver/metabolism* ; Male ; Rats ; Triglycerides/metabolism*

OBJECTIVESTo investigate the effects of glucose and free fatty acids (FFAs) on the proliferation and cell cycle of human vascular endothelial cells in vitro, and to examine whether the combined presence of elevated FFAs and glucose may cross-amplify their individual injurious effects.

METHODSCultured human vascular endothelial cells (ECV304) were incubated with various concentrations of glucose and/or FFAs (palmitate and/or oleate) for 24 - 96 h. Morphologic alterations were observed using a phase contrast microscope and an electron microscope. Inhibition of proliferation was measured by a colorimetric 3-[4, 5-dimethyl thiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) assay. Cell viability was determined using trypan blue exclusion. Distribution of cells along phases of the cell cycle was analyzed by flow cytometry.

RESULTSGlucose 15 or 30 mmol/L, palmitate (PA) 0.25 or 0.5 mmol/L, and oleate (OA) 0.5 mmol/L inhibited proliferation and accelerated death of endothelial cells in a dose-and-time-dependent manner. After treatment with elevated glucose and/or FFAs, the G(0)/G(1) phase cells increased, whereas S phase cells decreased, suggesting that high glucose and/or FFAs mainly arrested endothelial cells at G(0)/G(1) phase. The inhibitive rates of proliferation and population of dead cells in endothelial cells incubated with glucose plus FFAs (glucose 30 mmol/L + PA 0.25 mmol/L, glucose 30 mmol/L + OA 0.5 mmol/L, glucose 30 mmol/L + PA 0.25 mmol/L + OA 0.5 mmol/L) increased more markedly than those treated with high glucose or FFAs (PA and/or OA) alone.

CONCLUSIONBoth high ambient glucose and FFAs can inhibit proliferation and accelerate death of endothelial cells in vitro. These changes were cross-amplified in the combined presence of high levels of glucose and FFAs.

Cell Division ; drug effects ; Cell Survival ; drug effects ; Cells, Cultured ; Endothelium, Vascular ; cytology ; Fatty Acids, Nonesterified ; pharmacology ; Glucose ; pharmacology ; Humans

OBJECTIVETo establish an in vitro cell model for investigating hepatic steatosis in non-alcoholic fatty liver disease.

METHODSL-02 cells cultured in 1640 containing 10% fetal bovine serum were divided into control group and model group. At 70%-80% confluency, L-02 cells in the model group were exposed to a long-chain mixture of free fatty acids (FFA, oleate and palmitate ) for 24 h, and cells in control group were treated with fresh medium. Lipid droplets in the cells were observed and total lipid content was determined with Oil Red O staining. The morphology of lipid droplets, trilyceride level, malonaldehyde content and cell apoptosis rate were evaluated to verify the cell model, and the effect of Huganqingzhi tablet on the lipid droplets was observed.

RESULTSA large number of lipid droplets were found in the cell model, which showed markedly increased level of triglyceride without significant changes of malonadehyde content or cell apoptosis rate. Intervention with two doses of Huganqingzhi tablet significantly decreased the number of lipid droplets and trilyceride content in the cell model.

CONCLUSIONhepatic steatosis L-02 cell model can be established by long-chain mixture of free fatty acids (oleate:spalmitate=2:1) for therapeutic drug studies.

Apoptosis ; Cell Line ; Drugs, Chinese Herbal ; pharmacology ; Fatty Acids, Nonesterified ; chemistry ; Fatty Liver ; Humans ; Malondialdehyde ; analysis ; Non-alcoholic Fatty Liver Disease ; Triglycerides ; analysis

To evaluate the roles of hyperglycemia and increased plasma FFA level in the development of insulin resistance, we examined the effects of phlorizin and acipimox treatments on tissue sensitivity to insulin in streptozotocin(STZ)-diabetic rats. Insulin sensitivity was assessed with the glucose-insulin clamp technique. Blood glucose concentration was clamped at basal levels of control and diabetic states, and plasma insulin concentrations were clamped at the levels of basal, approximately 60 and approximately 1500 microU/ml. In diabetic rats, the basal blood glucose and plasma FFA levels in the fasting state were elevated, while the plasma insulin concentration was lower than in normal controls. Moreover, diabetic rats became glucose intolerant after intravenous injection of glucose. The metabolic clearance rate(MCR) of glucose showed a decrease of basal and insulin stimulated response in diabetic rats. As results of the glucose-insulin clamp study and intravenous glucose tolerance test, insulin resistance was developed in STZ-diabetic rats. Phlorizin treatment of diabetic rats recovered insulin sensitivity to nearly normal levels and improved glucose tolerance, but had no effect on insulin action in controls. Insulin sensitivity was also improved by acipimox treatment in diabetic rats, but did not reach normal levels. These results show that hyperglycemia is an obvious causative factor of insulin resistance, and increased FFA level may also act on the development of insulin resistance in STZ-diabetic rats.
Animal ; Antilipemic Agents/*pharmacology ; Blood Glucose/analysis ; Diabetes Mellitus, Experimental/*metabolism ; Fatty Acids, Nonesterified/blood ; Female ; *Insulin Resistance ; Phlorhizin/*pharmacology ; Pyrazines/*pharmacology ; Rats ; Rats, Sprague-Dawley ; Streptozocin

To examine the role of glycogen synthase kinase 3 (GSK-3) in the apoptosis of pancreatic beta-cells to better understand the pathogenesis and to find new approach to the treatment of type 2 diabetes, apoptosis was induced by oleic acid (OA) in INS-1 cells and the activity of GSK-3 was inhibited by LiCl. The PI staining and flow cytometry were employed for the evaluation of apoptosis. The phosphorylation level of GSK-3 was detected by Western blotting. The results showed that OA at 0.4 mmol/L could cause conspicuous apoptosis of INS-1 cells and the activity of GSK-3 was significantly increased. After the treatment with 24 mmol/L of LiCl, a inhibitor of GSK-3, the OA-induced apoptosis of INS-1 cells was lessened and the phosphorylation of GSK-3 was increased remarkably. It is concluded that GSK-3 activation plays an important role in OA-induced apoptosis in pancreatic beta-cells and inhibition of the GSK-3 activity can effectively protect INS-1 cells from the OA-induced apoptosis. Our study provides a new experimental basis and target for the clinical treatment of type-2 diabetes.
Apoptosis/*drug effects ; Cell Line ; Fatty Acids, Nonesterified/*pharmacology ; Glycogen Synthase Kinase 3/*metabolism ; Insulin-Secreting Cells/*cytology ; Oleic Acid/pharmacology ; Phosphorylation

This study sought to assess effect of high level of glucose or free fatty acids (FFAs) on apoptosis of vascular endothelial cell, and to examine whether the combined presence of high concentrations of glucose and FFAs may cross-amplify their individual detrimental effect. Cultured endothelial cell sline (ECV304) were incubated with various concentrations of glucose and/or selected FFAs (palmitate and/or oleate) for 24-96 h. Microscopic changes were observed by electron microscopy. DNA fragmentation was visualized by agarose gel electrophoresis. Apoptotic percentage of endothelial cells were determined using flow cytometry. After treatment with 30 mmol/L of glucose, 0.25 of palmitate (PA) and 0.5 mmol/L of oleate (OA) respectively, typical apoptosis morphological changes including condensed chromatin, nuclear fragmentation, reduction in volume and apoptotic bodies were observed under electron microscope, and the agarose gel electrophoresis of DNA from cells treated with high levels of glucose and FFAs revealed "Ladder" pattern. Both high concentrations of glucose and FFAs elicited apoptosis in a time- and dose-dependent manner in endothelial cells, and flow cytometry showed the cells were mainly blocked at G0/G1 phase. The apoptotic percentage of endothelial cells treated with high levels of glucose plus FFAs was significantly higher than that treated with high concentration of glucose or FFAs (PA and/or OA) alone. Both glucose and FFAs at high concentrations can induce apoptosis of endothelial cells. The combined presence of high level of glucose and FFAs may cross-amplify their individual effect.
Apoptosis ; drug effects ; Cells, Cultured ; Dose-Response Relationship, Drug ; Endothelial Cells ; cytology ; Fatty Acids, Nonesterified ; pharmacology ; Glucose ; pharmacology ; Humans ; Umbilical Cord ; cytology

BACKGROUNDPeroxisome proliferator-activated receptor (PPAR) alpha is one of the subtypes of PPARs. It regulates metabolism of lipid and lipoprotein, as well as glucose homeostasis. In addition, PPARalpha influences cellular proliferation, inflammation, differentiation and apoptosis, which plays a vital role in cardiovascular diseases. The purpose of this study was to investigate the role and mechanisms of PPARa activation in relation to acute myocardial damage induced by isoproterenol in rats.

METHODSThirty male Wister rats were randomly divided into control group, isoproterenol (Iso) injured group and fenofibrate (FF) treatment group. Acute myocardial damage caused by isoproterenol intraperitoneal injection induced ischemia was established. We determined the levels of creatine kinase (CK) and lactic dehydrogenase (LDH) in serum as well as the concentrations of free fatty acids (FFA) in serum and myocardium. The mRNA expressions of PPARa, muscular type carnitine palmitransferase (M-CPT-I) and medium chain lipid acetyl coenzyme A dehydrogenase (MCAD) were analyzed by reverse transcription-polymerase chain reaction (RT-PCR).

RESULTSCompared with the control group, the levels of serum CK and LDH were significantly increased after FF and Iso treatments. Moreover, the concentrations of FFA in both serum and myocardium were obviously increased in the Iso group and FF group, while the mRNA expressions of PPARalpha, M-CPT-I and MCAD declined, respectively (P < 0.01). When compared with the Iso group, significant decreases in serum CK and LDH were observed in the FF group. The concentrations of FFA both in serum and myocardial tissue were markedly decreased in the FF group, while the expressions of PPARalpha, M-CPT-I and MCAD mRNA were increased (vs. Iso, P < or = 0.01).

CONCLUSIONSThe utilization of FFA was reduced in isoproterenol induced acute myocardial damage. PPARalpha activation by its activator fenofibrate may play a key role in energy metabolism in acute myocardial damage induced by isoproterenol in rats.

Animals ; Creatine Kinase ; blood ; Energy Metabolism ; Fatty Acids, Nonesterified ; metabolism ; Fenofibrate ; pharmacology ; Heart ; drug effects ; Isoproterenol ; toxicity ; L-Lactate Dehydrogenase ; blood ; Male ; PPAR alpha ; physiology ; Rats ; Rats, Wistar

BACKGROUNDPreeclampsia (PE) is associated with abnormal fatty acid beta-oxidation (FAO), especially metabolic disorders of long-chain fatty acid oxidation. The role of FAO dysfunction in inadequate invasion is unclear. The aim of this study was to explore the influence of various lengths fatty acids oxidation on invasiveness of trophoblasts.

METHODSPrimary human trophoblast cells and HTR8/SVneo cells were treated with fatty acids of various lengths. Morphological changes, lipid deposition and ultrastructure changes of trophoblast cells were detected. Cells invasiveness was determined by transwell insert. CPT1, CPT2 and long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) protein expression were analyzed. The correlation between intracellular lipid droplets deposition and cells invasiveness was evaluated.

RESULTSCells treated with long-chain fatty acids showed significant increased lipid droplets deposition, severe mitochondrial damage, decreased CPT2 and LCHAD protein expression (P < 0.05) but no significant difference in CPT1 protein expression (P > 0.05). Invasiveness of the trophoblast cells of the LC-FFA group significantly decreased (P < 0.05). Intracellular lipid droplets deposition was negatively correlated with invasivenss (R = -0.745, P < 0.05).

CONCLUSIONTrophoblast cells after stimulation with long chain fatty acids exist fatty acid oxidation disorders, and reduce the ability of trophoblastic invasion.

Cell Line ; Cells, Cultured ; Fatty Acids, Nonesterified ; pharmacology ; Humans ; Lipid Metabolism ; Long-Chain-3-Hydroxyacyl-CoA Dehydrogenase ; metabolism ; Trophoblasts ; drug effects

OBJECTIVETo observe the intervention and mechanism of Qushi Huayu Recipe (QHR) on gene expression profiles in high lipid diet induced fatty liver rats.

METHODSFatty liver model was prepared in 20 male SD rats using single high fat diet (88% common forage +2% cholesterol +10% lard). Four weeks after modeling they were divided into the model group and the QHR group according to random digit table, 10 in each group. QHR (at 0. 93 g crude drug/100 g body weight) and distilled water was respectively to rats in the QHR group and the model group by gastrogavage while modeling, once per day. Meanwhile, 10 SD male rats were recruited in a normal group, administered with equal volume of distilled water by gastrogavage. At the end of week 8 all rats were sacrificed, and blood and livers were collected for subsequent analysis. Contents of liver triglyceride (TG) and free fatty acid (FFA) , activities of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were detected using biochemical assay. Pathological changes of liver tissue were observed using H&E and oil red O stain. Liver gene expressions were detected by Affymetrix gene expression profiles. Differentially expressed genes were compared between the QHR group and the model group, functions of differentially expressed genes and signal pathways involved analyzed. Ten differentially expressed genes involved in glycolipid metabolism with fold change more than 2 were selected for verification by real-time PCR.

RESULTS(1) Compared with the normal group, contents of liver TG and FFA, and serum activities of ALT and AST obviously increased in the model group (P <0. 01). Compared with the model group, contents of liver TG and FFA, and activities of ALT and AST obviously decreased in the QHR group (P <0. 05, P <0. 01). QHR could reduce high fat induced fatty degeneration of liver cells , alleviate inflammation, and improve pathological changes of liver tissue. (2) Compared with the model group, there were 80 differentially expressed genes (with fold change > 2, P < 0.05) with clear functions and appointed gene names, including 44 up-regulated and 36 down-regulated genes. Eighty genes were involved in 27 signal pathways with statistical difference, including glycerolipid metabolism, adipocytokine signaling pathway, insulin signal pathway, drug metabolism signal pathway, etc (P < 0.05). (3) RT-PCR results of 10 glycolipids metabolism regulating genes such as Gk, Scd1, Gpat2, G6pc, Irs1, and so on showed that all RT-PCR genes were completely coincide with up-regulated or down-regulated tendency in results of gene chips. 80% genes had approximate fold change.

CONCLUSIONQHR could regulate gene expressions related to fat metabolism, carbohydrate metabolism, anti-lipid peroxidation, and drug metabolism in high fat diet induced fatty liver rats, and its comprehensive pharmacological actions could be manifested.

Alanine Transaminase ; metabolism ; Animals ; Aspartate Aminotransferases ; metabolism ; Carbohydrate Metabolism ; Diet, High-Fat ; Drugs, Chinese Herbal ; pharmacology ; Fatty Acids, Nonesterified ; metabolism ; Fatty Liver ; metabolism ; Lipid Metabolism ; Lipid Peroxidation ; Male ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Transcriptome ; drug effects ; Triglycerides ; metabolism