| Objective：Diabetes is becoming a serious public health problem in the world.Insulin resistance (IR) characterized by insulin signaling defection is the root cause ofthe onset of type2diabetes mellitus (T2DM). The liver is one of the major target organsof insulin action, and is also the primary organ to maintain the glucose homeostasis. Thehepatic IR plays an important role in the cause and development of T2DM. Palmiticacid(PA) belongs to saturated fatty acid which can induce hepatic IR, but the exactmechanisms have not been known. The amount of evidence shows that mitochondrialdysfunction is one the important mechanism of these causes. Mitochondria are the mainplace of ATP synthesis and fatty acid oxcidates. It can produce lots of hydrogen carrierswhich then join in electron transporting respiratory when overwhelming fatty acids.Thus the mitochondria redox balance would be damaged and resulted in mitochondrialenergy generation disorder, and thereby causes a massive release of intracellularreactive oxidate species (ROS). Meanwhile, ROS is not only a second messenger, but itis also an important factor to trigger intracellular oxidative stress acting on the proteinsand lipids, thereby causing the change in hormonal signals, glucose and lipidmetabolism disorders including insulin resistance. However, the details about changingof mitochondrial function and insulin signaling during the process of PA-inducedhepatic insulin resistance is still in learning. So, this study is aimed to establlishPA-induced IR modle in L02cells, observe the dynamic changes in mitochondrialfunction and insulin signaling pathway, explore the key mechanism in PA-inducedhepatic IR and find a new target of treatment and intervention of IR.Methods：The human hepatocyte L02cells were normally cultured. We choseLogarithmic growth phase cells which were divided into the following groups：0.5%BSA group, PA0.125mmol/L group, PA0.25mmol/L group, PA0.5mmol/L group. Besides, it contains blank group in the sample. Using MTT assay to test lipotoxicityafter the sample was exposed to PA for24h. then, According to the results，we selectedthe PA0.25mM to incubate logarithmic growth phase cells，which were randomlydivided into control group, PA8h group, PA16h group, PA24h group. After that，thecells were dealed with the following items.(1) Cellular glycogen contents weremeasured by anthrone after incubating in the presence or absence of insulin100nM for3h.(2) glucose consumption was assessed by Glucose oxidase kit after incubating in thepresence or absence of insulin100nM for3h.(3) lipid accumulation was detected by oilred O staining.(4) Intracellular ATP content was detected by the Biochemistrychemiluminescence, Cellular ROS generation and mitochondrial membranepotential(MMP) was tested by Fluorospectrophotometry.(5) Western Blot was used toanalyze the PKCε mitochondrial translocation and UCP2, phosphorylation of signalmolecules such as JNK, IRS1and AKT.Results：(1) The difference of cell viability among different concentrations of PA treatmentCompared with the control group, the low PA concentration0.125mM and0.25treated on L02hepatocytes for24h didn’t show any difference in cell viability; L02hepatocytes treated with PA for24h decreases cell viability. It Suggests that lowconcentrations of PA has no effect on cell survival.(2) The effect of insulin sensitivity and lipid accumulation on PA-induced cellsIn the basic state, there is no significant difference of glycogen synthesis andglucose consumption among the groups. Compared to the control group without insulin,the control group with insulin has significant increased glycogen synthesis and glucoseconsumption. The L02cells were treated with PA for8h and16h, In theinsulin-stimulated group, the glycogen synthesis and glucose consumption increasedsignificantly compared to the control group without insulin. But the quantity did notincrease as same as control group with insulin. With the increasing of PA treatment timethe hepatic insulin resistance was decreased. PA is induced to hepatocytes for24h, butthere is no difference of glycogen synthesis and glucose consumption between PA24hwith insulin and control group without insulin. Oil Red O staining showed that therewas no lipid accumulation in control group. But little amount of lipid accumulation inPA0.25mM is seen in8h group. As the PA incubation time increases, the cells increasethe lipid accumulation. But in PA24h group there is lot of lipid accumulation. (3) Changes of mitochondrial functionMitochondrial function indicators showed that the ROS generation wassignificantly increased at PA8h(P<0.001) compared to the control group. IntracellularROS levels increases at the time of PA incubation, the ROS increased drastically aftertreatment for24hours with PA (P<0.001). Within this range, the increasing ofPA-induced ROS generation showed time-dependent manner. Compared with controlgroup, cellular ATP synthesis and MMP did not change in PA8h group, The ATP andMMP were reduced in PA16h group, In the PA24h group both of these two indicatorsdecreased more significantly. These results indicate that ROS changes earlier than theother indicators mitochondrial functions.(4) Changes of mitochondrial PKCε and UCP2levelCompared with control group, in PA8h, the PKCε mitochondrial translocation wasincreased, But the difference has no significance at this moment. Mitochondrial PKCεprotein level increased significantly in the PA16h group and24h group. Compared withcontrol group, mitochondrial UCP2protein content was increased significantly at8h.After PA treated for16h and24h, The UCP2content was decreased compared to thecontrol group, and the UCP2content in16h and24h has no significant differencecompared to the control group.(5) Changes of insulin signaling pathway proteins phosphorylation.Compared with control group, in PA8h group, JNK phosphorylation levelsincreased significantly (P <0.05); JNK phosphorylation levels changes with the PAtreatment time. In PA8h group the JNK phosphorylation increased more significantlycompared with control group. The phosphorylation of the IRS-1Ser307was detected inPA24h compared with control group. The insulin-stimulated AKT Ser473phosphorylation level decreased in PA8h group. There is no difference among8h,16hand24h at AKT Ser473phosphorylation. The results prompted that there is arelationship between JNK phosphorylation changing and insulin signal phosphorylationchanging.Conclusion：(1) L02hepatocytes treated with PA for24h induces insulin resistance.(2) In the process of PA-induced L02hepatocytes IR, The increase of ROS generationsignificantly in the beginning causes reducing of mitochondrial function andincrease of phosphorylation of JNK, thereby by causing IR. Thus it indicated that ROS may be the trigger of PA-induced L02hepatocytes IR.(3) In the process of PA-induced L02hepatic IR, ROS induces PKCε mitochondrialtranslocation. |
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