The Effect Of4-hydroxyisoleucine On The Insulin Resistance And Its Molecular Mechanism In3T3-L1Adipocytes

PartⅠ4-HIL demonstrates no cytotoxic effects on3T3-L1cellsand promotes glucose uptakeBackground and Objective: Chinese medicine fenugreek (Trigonellafoenum-graecum) has been confirmed in many studies that it can efficiently lowerblood glucose and correct dyslipidemia with good safety. So it is widely used in theclinical work.4-Hydroxyisoleucine (4-HIL) is considered to be one of the mostimportant ingredients of fenugreek with biological activity. We intended to observethe cytotoxic effects of4-HIL on3T3-L1cells and whether4-HIL alleviated insulinresistance (IR) in3T3-L1adipocytes.Methods: MTT assay was performed to determine the drug safety of4-HIL. Then3T3-L1adipocytes were incubated in25mmol/L glucose and0.6nmol/L insulin toinduce IR followed by addition of4-HIL at different concentrations.2-deoxy-[3H]-D-glucose (2-DOG) method was used for assessing glucose uptakerate.Results:4-HIL at the concentration range of0~200μmol/L presents no cytotoxiceffects on3T3-L1cells. After stimulation by glucose and insulin for18h，2-DOGuptake rate in3T3-L1adipocytes decreased by67%, which was reversed by4-HIL.Conclusion:4-HIL is safe and improves IR in3T3-L1adipocytes. PartⅡ4-HIL down-regulates protein expression of TACEand up-regulates TIMP3in insulin-resistant3T3-L1adipocytesBackground and Objective: IR is know as a chronic low-grade inflammatorydisease. The adipose tissue produces excessive cytokines, such as tumor necrosisfactor (TNF-α), which play a negative effect on the insulin-signal-transductionresulting in exacerbation of IR. Such biological active TNF-α is usually referred tothe soluble TNF-α (sTNF-α), which is cleaved from the transmembrane form(Tm-TNF-α) by TNF-α converting enzyme (TACE) in human body. The naturalinhibitor of TACE is tissue inhibitor of metalloproteinase (TIMP3) in vivo.Therefore, the reduction of sTNF-α through decreasing the expression of TACEand/or increasing the expression of TIMP3could improve IR. We intended toinvestigate the effects of4-HIL on the regulation of TACE and TIMP3proteinexpression and on the secretion of sTNFRⅠ in insulin-resistant3T3-L1adipocytes.Methods: The insulin-resistant3T3-L1adipocytes were treated with either4-HIL atdifferent concentrations or10μmol/L Pioglitazone (PIO), the later was taken aspositive control. Western-blot was performed to determine the protein expression ofTACE and TIMP3, the levels of sTNFRⅠ in cell supernatant were detected byELISA kit.Results:4-HIL significantly down-regulated TACE protein expression andup-regulated TIMP3protein expression in a dose-dependent manner ininsulin-resistant3T3-L1adipocytes（P<0.05）.4-HIL reduced the over-secretion ofsTNFRⅠ in a dose-dependent manner as well（P<0.001）.Conclusion:4-HIL improves IR in3T3-L1adipocytes through the regulation ofTACE/TIMP3protein, which thereby changes the proportion of sTNF-α andTm-TNF-α. Part Ⅲ The effect of4-HIL on insulin signaling pathway ininsulin-resistant3T3-L1adipocytesBackground and Objective:4-HIL was successfully extracted from the seeds offenugreek during the1970s. At present the possible mechanisms underlying itsglucose-lowering effect are linked to inhibition of glucose absorption,enhancement of insulin synthesis and secretion as well as activation of insulinsignaling pathway. We further intended to explore the impact of the4-HIL on theimportant molecules of insulin-signal-transduction pathway in3T3-L1adipocytes.Methods: The insulin-resistant3T3-L1adipocytes were treated with differentconcentrations of4-HIL. The protein expression of IRS-1, Akt and GLUT4wasinvestigated by western-blot. And then the phosphoserine protein expression ofIRS-1(P-IRS-1Ser307, P-IRS-1Ser318) and Akt (P-Akt Ser473) were alsoanalyzed.Results: The expression of IRS-1and GLUT4proteins in insulin-resistant3T3-L1adipocytes were increased after exposure to4-HIL（P<0.05）. While the proteinexpression of P-IRS-1Ser307and P-Akt Ser473were decreased（P<0.05）. Therewas no significant change on Akt or P-IRS-1Ser318production.Conclusion:4-HIL relieves the inhibitory effect of sTNF-α on insulin signalingpathway to improve insulin sensitivity and thereby ameliorate obesity-linked IR.