Effects Of KLF14 Overexpression On Hepatic Insulin Resistance And The Relative Signal Pathway

PART ONEKLF14 MRNA AND PROTEIN EXPRESSION IN DIFFERENT TISSUES OF MOUCE AND PEOPLEObjective To observe the expression of KLF14 mRNA and protein in C57BL/6J, HFD-C57BL/6J, db/db mice and normal subjects, T2DM patients.Method Total RNA and protein of liver, adipose tissues, skeletal muscles in mice and adipose tissues, skeletal muscles in normal subjects and T2DM patients were extracted. Total RNA was reverse transcribed into cDNA, and mRNA expression of KLF14 in different tissues was detected by use of real-time PCR. Western blot was used to identify the protein expression of KLF14 in the tissues.Results Compared with C57BL/6J mice, KLF14 mRNA and protein in liver, adipose tissues and skeletal muscles were significantly decreased in HFD-C57BL/6J and db/db mice (P<0.01). Furthermore, KLF14 mRNA and protein in adipose tissues and skeletal muscles were also significantly reduced in T2DM patients (P<0.01).Conclusions KLF14 mRNA and protein were significantly decreased in HFD-C57BL/6J, db/db mice and T2DM patients, which indicate that KLF14 might play an important role in the metabolic disorders.PART TWO EFFECTS OF KLF14 OVEREXPRESSION ON THE KEY SIGNALING MOLECULES IN INAULIN SIGNAL PATHWAYObjective To determine the effects of overexpression of KLF14 on the key signaling molecules in PI3K/Akt signal pathway.Methods The recombinant plasmid expressing KLF14 (pIRES2-KLF14) was used to up-regulated the expression of KLF14 in hepal-6 cells. Effects of KLF14 on glucose uptake was assessed by 2-[3H]-deoxyglucose (2-DG) uptake. Western blot was used to identify the expression of p-InsR, InsR, p-IRSl, IRS1, p-Akt, Akt, p-GSK3p and GSK3β.Results Overexpression of KLF14 enhanced insulin-stimulated glucose uptake and the phosphorylation of insulin receptor (InsR), insulin receptor substrate-1 (IRS1), glycogen synthase kinase-3β (GSK3β) and Akt (P<0.05 or P<0.01). However, KLF14’s ability to increase glucose uptake and activation of Akt, GSK3β was significantly abolished by LY294002, a PI3K inhibitor (P< 0.01).Conclusion KLF14 overexpression might increase insulin sensitivity through the activation of PI3K/Akt signal pathway.PART THREEEFFECTS OF KLF14 OVEREXPRESSION ON THE IMPROVEMENT OF HEPATIC INSULIN RESISTANCE AND ITS POTENTIAL MECHANISMSObjective To identify the effect of KLF14 overexpression on the improvement of hepatic insulin resistance and its potential mechanism.Methods The insulin resistance cell model was constructed by incubating with high insulin and (or) glucose for 24 hr. MTT asssy was used to detect the cell viability. The up-regulation of KLF14 in hepal-6 cells was obtained by transfection of pIRES2-KLF14.2-[3H]-deoxyglucose (2-DG) uptake assay was used to detected the glucose uptake in hepal-6 cells. The expression of p-Akt, Akt was assessed by western blot.Results Hepal-6 cells cultured in medium with high insulin (10"8M) or high glucose (25mM) exhibited a state of insulin desensitization characterized by decreasing 2-DG uptake and Akt phosphorylation when stimulated by insulin (both P<0.01). However, treatment with pIRES2-KLF14 in hepal-6 cells prevented high insulin and/or high glucose-induced decrease in 2-DG uptake and Akt phosphorylation (P<0.05 or P<0.01). The ability of KLF14 was significantly attenuated by LY294002 (P<0.05 or P<0.01). In addition, for serum treat experiment, in insulin-stimulated state,2-DG uptakes and Akt phosphorylation were significantly decreased in hepal-6 cells incubated with T2DM serum (p<0.05 or P<0.05). However, pIRES2-KLF14 treatment prevented T2DM serum-induced decrease in 2-DG uptake and Akt phosphorylation in these cells (both P<0.05). Importantly, the PI3K inhibitor LY294002 could also block KLF14-induced increase in 2DG uptake and Akt phosphorylation in cells incubated with normal or T2DM serum (P<0.05 or P<0.01).Conclusions KLF14 gene overexpression may play a great role in increasing the insulin sensitivity and improving the insulin resistance through the PI3K/Akt signal pathway.