The Regulatory Mechanism Of NEFAs On Glucolipid Metabolism In Bovine Hepatocytes In Vitro

Because of the dry matter intake decreased and the energy requirement increased,dairy cows often in a state of negative energy balance, then initate lipid mobilizationand lead to high serum β-hydroxybutyrate and high non-esterified fatty acid, this caninduce ketosis and fatty liver in dairy cows. There have studies indicated that dairycows with ketosis or fatty liver offen along with insulin resistance and inflammation,however, insulin is closely related to glucose and lipid metabolism. In addition, theactivation of NF-κB inflammatory signaling pathway is also closely related to insulinresistance. Stydies in obese or high-fat diet mice demonstrated that NEFAs wasclosely related to inflammatory and insulin resistance. As we all have known thatTLRs is a class of pathogen recognition receptor, it can activate NF-κB signalingpathway, and promote the transcriptions of pro-inflammatory factor. There havestudies indicated that inflammatory factor can influence the transmition of insulinsignaling pathway to downstream by different pathway to induce serine residuephosphorylation of insulin receptor substrate, this can reduce the physiologicalfunction of insulin and result in insulin resistance. There have studies indicated thatNEFAs could activate TLR4in vitro or vivo. However, there have no studies on therelationship of high-dose NEFAs and TLR4/NF-κB inflammatory signaling pathwayand liver insulin resistance, as well as how NEFAs cause insulin resistance. In thisstudy, we cultured bovine hepatocytes in vitro to discuss the relationship and theinduction mechanism of TLR4/NF-κB inflammatory signaling pathway and liverinsulin resistance. In addition, studies indicated that NEFAs as a signal molecule canregulate lipid metabolism of hepatocytes directly. PPARα is the most importanttranscription factor in regulating lipid oxidation. SREBP-1c and ChREBP areimportant transcription factors in regulating lipid synthesis. Consequently, we studiedthe influences of high NEFAs on the three nuclear transcription factors by fatty liversheep in vivo and hepatocytes in vitro. Studies indicate that NEFAs increased the expression of TLR4and the number ofP65that into the nuclears in bovine hepatocytes, activated NF-κB inflammatorysignaling pathway, at the same time, the concentration in hepatocytes medium and themRNA level of IL-6and TNF-α were increased. The inhibitor of TLR4decreased thenumber of P65into nuclears and the expressions of IL-6and TNF-α, suppressed theinflammatory response caused by NEFAs. These results indicated that NEFAs couldactivate TLR4/NF-κB inflammatory signaling pathway. In addition, we found thatNEFAs increased the phosphorylation level of IRS1, decreased the phosphorylationlevel of AKT and the protein level of PI3K, inhibited the insulin signaling pathway,cause insulin resistance. The inhibitor of NF-κB decreased the phosphorylation levelof IRS1, increased phosphorylation level of AKT and the protein level of PI3K,alleviate the inhibiting effects of NEFAs on insulin pathway. These results indicatethat NEFAs influences insulin pathway by NF-κB inflammatory signaling pathway,and we infer that inflammation can cause insulin resistance in vitro.Ketosis and fatty liver are common nutritional metabolic diseases in perinataldairy cows, and they are characterized by high NEFAs and high BHBA. SREBP-1c,ChREBP and PPARα are key transcriptional factors in lipid metabolism. We addedhigh-dose NEFAs to bovine hepatocytes, we found that high-dose NEFAs increasedthe protein and mRNA levels of SREBP-1c and ChREBP which are key genes in lipidsynthesis, the number into nuclears also increased, and their downstream genes ACCαand FAS are accordance with SREBP-1c and ChREBP. However, the protein level andnumber into nuclears of PPARα were obviously decreased in high-dose NEFAs group,the mRNA levels of its downstream genes L-FABP and ACSL-1were also decreased.These results indicated that NEFAs increased the expression levels of lipid synthesisgenes and decreased the expression levels of lipid oxidation genes. In order to verifythe role of NEFAs on lipid deposition, we established the model of fatty liver of sheep.We found that the serum level of NEFAs in fatty liver groups was obviously higherthan that in healthy groups. The protein levels of SREBP-1c and ChREBP were higherthan that in healthy group, but no change of PPARα. These shown that high-doseNEFAs increased lipid synthesis and lower lipid oxidation, and at last caused lipid deposition in liver.In conclusion, all of these results indicate that high-dose NEFAs can activateTLR4/NF-κB inflammatory signaling pathway, reduce the sensitivity of insulinpathway. In addition, high-dose NEFAs can accelerate the lipid synthesis and inhibitelipid oxidation, and at last cause lipid deposition in liver.