The Improvement Effect And Underlying Mechanism Of Casein Glycomacropeptide-derived Peptides On Hepatic Insulin Resistance

Currently,obesity,insulin resistance and related metabolic disorders have become a serious public health problem,which seriously threatens human health.Our previous study showed that casein glycomacropeptide hydrolysates obtained by papain(GHP)possessed higher antioxidant and anti-inflammatory activity compared with intact casein glycomacropeptide.However,the improvement effects of GHP on hepatic insulin resistance remained unclear.In current study,HepG2 cells and C57BL/6J mice were used to evaluate the improvement effect and underlying mechanism of GHP on hepatic insulin resistance in vitro and in vivo.The main results and conlusion in present study were as follows:1.GHP increased cellular glucose uptake and intracellular glycogen in insulin-resistant HepG2 cells,which contributed to the improvement of insulin resistance.The serum lipids in HFD mice,including TG,TC,FFA,HDL-c and LDL-c were reduced by GHP treatment.GHP also reduced the serum fasting blood glucose and insulin in HFD mice.Moreover,the serum inflammatory factors,such as TNF-?,IL-6,MCP-1 and leptin and liver enzyme markers ALT and AST in HFD mice were decreased by GHP.GHP reduced body weight,and lipid accumulation in liver and adipose tissue of HFD mice.GHP improved glucose tolerance and increased insulin sensitivity in HFD mice.These results suggested that GHP improved hepatic insulin resistance.2.GHP inhibited IRS-1 Ser phosphorylation and increased Akt phosphorylation in liver tissue of HFD mice,which contributed to the regulation of GHP on phosphorylation of key glycogenic proteins(GSK3? and GS)and intracellular glycogen.These improvement effects of GHP on hepatic insulin resistance were associated with the increased phosphorylation of AMPK and the decreased phosphorylation of MAPK in liver tissue of HFD mice.Moreover,GHP inhibited the protein expression levels of PPARy,C/EBPa and FAS,lipid accumulation in adipose tissue and inflammatory response in HFD mice,which were involved in the increased phosphorylation of AMPK and the inhibition of MAPK phosphorylation and NF-?B signaling activation.These beneficial effects of GHP on adipose tissue contributed to improve hepatic insulin resistance.3.The peptide IPPKKNQDKTE was identified from GHP.The expression levels of key gluconeogenic enzymes(G6Pase and PEPCK)were inhibited by the peptide IPPKKNQDKTE in insulin-resistant HepG2 cells.GHP also regulated the phosphorylation of key glycogenic proteins(GSK3? and GS).These improvement effects of GHP on insulin-resistant HepG2 cells contributed to increase cellular glucose uptake and intracellular glycogen.The improvement mechanisms of GHP on insulin resistance in HepG2 cells included two aspects:(1)The peptide IPPKKNQDKTE promoted the phosphorylation of AMPK and Akt in insulin-resistant HepG2 cells,and inhibited IRS-1 Ser phosphorylation.Knockdown AMPK using siRNA in HepG2 cells reversed the regulation of the peptide IPPKKNQDKTE on the phosphorylation of IRS-1 Ser and Akt.These results suggested that the peptide IPPKKNQDKTE improved insulin signaling transduction and insulin resistance via increasing the phosphorylation of AMPK in insulin-resistant HepG2 cells.(2)The peptide IPPKKNQDKTE decreased the phosphorylation of MAPK in insulin-resistant HepG2 cells,which contributed to inhibit IRS-1 Ser phosphorylation,increase cellular glucose uptake and improve insulin resistance.Moreover,the peptide IPPKKNQDKTE reduced the levels of ROS in insulin-resistant HepG2 cells.NAC,a ROS scavenger decreased the phosphorylation of MAPK,which indicated that the peptide IPPKKNQDKTE decreased the phosphorylation of MAPK by reducing the levels of ROS in insulin-resistant HepG2 cells.Additionally,IPPKKNQDKTE increased Nrf2 nucleus translocation and induced HO-1 expression.HO-1 inhibitor and activator regulated IPPKKNQDKTE-mediated suppression on the level of ROS in insulin-resistant HepG2 cells,which suggested that the ROS reduction by IPPKKNQDKTE in insulin-resistant HepG2 cells was mediated by Nrf2/HO-1 signaling.Taken together,GHP and GHP-derived peptide IPPKKNQDKTE improved IRS-1/Akt signaling transduction and hepatic insulin resistance via activating AMPK phosphorylation and inhibiting MAPK phosphorylation.