Effects Of TLR4on Fatty Acid-induced Insulin Resistance Of Human Skeletal Muscle Cell

Objective: Metabolic disease is a worldwide problem, affecting people’squality of life seriously. The occurrence of metabolic diseases is associated withobesity caused by positive balance of long-term energy intake. When obesityleads to other metabolic diseases, the formation of systemic IR is an importantpart of it. Thus, we conclude that IR is the key to the occurrence of metabolicdiseases. Adipose tissue and IR induced in free fatty acids increase; thoseincreased in free fatty acids and long-term energy balance will induce obesity. Tosome extent, obesity is a low-grade inflammation. However, the mechanism ofobesity inducing inflammatory response is not clear. Body immune system isprecisely regulated, and inflammation occurrence tends to have one or severalkey adjustment points, which has been proved that TLR4may be a key regulationpoint of obesity caused by low-grade inflammatory response. TLR4plays an important role in the inflammatory factor gene promoter and transcription process,and also plays an important role in obesity caused by inflammation, but there isstill no exact evidence to support the above assumptions.In this experiment, we choose human skeletal muscle cells as an object,studying TLR4expression levels of inflammatory factors activity and insulinresistance, and fatty acids of TLR4activity and its downstream inflammatoryfactors.Methods:1. Human skeletal muscle cells were treated with palmitinic acid and DHA(100μmol/L、200μmol/L、400μmol/L), using MTT to test the reproductiveactivity4h，8h，12h，24h later. At the time of the24h hour, determine thequality of glucose in cell culture fluid with GOD-POD. Western Blot wasused to detect the expression of TLR4，MyD88，CRP; Real-time PCR wasused to detect the expression of TLR4，MyD88，CRP，TNF-α and IL-6mRNA.2. Human skeletal muscle cells were treated with LPS and PMB for24hours,then determine the quality of glucose in cell culture fluid with GOD-POD.Western Blot was used to detect the expression of TLR4，MyD88; Real-timePCR was used to detect the expression of TLR4，MyD88，CRP，TNF-α andIL-6mRNA.Results:1. With the treatment of palmitinic acid, the absorbtion of glucose was lowerthan control group markedly（P<0.05）; TLR4and the inflammatory factorsof its downstream such as TNF-α， IL-6， CRP, and its adaptormolecule—MyD88mRNA all expressed higher than control group（P<0.05）. While there is no significant difference statistically in the quality of protein.On the other hand, DHA treatment had no effects.2. Inflammatory factors such as TNF-α，IL-6，CRP and MyD88in TLR4highlyexpressed group were upper than in control group（P<0.05）, with theabsorption of glucose decreased significantly（P<0.05）. In TLR4lowlyexpressed group inflammatory factors such as TNF-α，IL-6，CRP andMyD88were lower than in control group（P<0.05）, while there is nosignificant difference statistically of the absorption of glucose decreasedsignificantly.Conclusion:Saturated fatty acid can induce release of inflammatory factors and decreasethe absorption of glucose in Human skeletal muscle cells, so lower the sensitivityof insulin to induce insulin resistance, and these effects were mediated byactivation of TLR4. while ω-3polyunsaturated fatty acid have no effects.