The Role Of Toll-like Receptor4/MyD88-mediated PI3K/Akt Signaling On The Regulation Of Hepatic Glucose And Lipid Metabolism In Mice

Objective Non-alcoholic fatty liver disease (NAFLD) is mainly attributed tohyperglycemia and hyperinsulinemia. Insulin signaling and glucose signaling play acritical role in regulating the expression of key enzymes for syntheses of fatty acids andtriglyceride (TG). Our preliminary study observed hepatic TG accumulation but thelevel of blood glucose decreased instead of increased when mice were injected a singledose of lipopolysaccharide (LPS), a natural ligand of toll-like receptor4(TLR4). Thus,we hypothesize that TLR4/MyD88signaling plays an important role in regulatinghepatic glucose and lipid metabolism. To explore the molecular mechanism throughwhich TLR4/MyD88-mediated PI3K/Akt signaling regulats hepatic glucose and lipidmetabolism, the present study was to compare differential effects of LPS on hepaticglucose and lipid metabolism between tlr4gene mutant mice and tlr4wild-type mice.Methods The present study included ten separate experiments. In experiment1, toinvestigate the effects of LPS on fasting blood glucose, C3H/HeJ (tlr4gene mutant),C3H/HeN and ICR (tlr4wild-type) mice were intraperitoneally (i.p.) injected with LPS(1.0mg/kg). Fasting blood glucose was examined at different time points (0,2,6,12and24h) after LPS administration. In experiment2, to explore the effects of LPS on theexpression of genes for gluconeogenesis, ICR mice were i.p. injected with LPS (1.0mg/kg). Liver was collected6h after LPS. The expression of hepaticglucose-6-phosphatase (g-6-pase) and phosphoenolpyruvate carboxylase (pepck) wasmeasured using real-time RT-PCR. In experiment3, to explore the effects of LPS onglucose tolerance and insulin tolerance, C3H/HeJ, C3H/HeN and ICR mice were i.p.injected with LPS (1.0mg/kg). GTT and ITT were performed12h after LPS. In experiment4, to explore the effects of LPS on hepatic Akt phosphorylation, C3H/HeJ,C3H/HeN and ICR mice were i.p. injected with LPS (1.0mg/kg). Liver sample wascollected at2,6and12h after LPS. Hepatic Akt phosphorylation was determined byWestern Blotting. In experiment5, to investigate the effects of LPS on hepatic insulinsignaling, ICR mice were randomly divided into four groups (Control, LPS group,Insulin alone group, and Insulin+LPS group). In Insulin alone and Insulin+LPS groups,mice were treated with insulin (2.0U/kg)6h after LPS. ICR mice were i.p. injectedwith LPS (1.0mg/kg). Liver sample was collected5min after insulin.IRS-1tyrosinephosphorylation was determined using Co-IP. The interaction between phosphorylatedIRS-1and P85was measured using Co-IP. Hepatic mRNA of IRS-1and IRS-2wasdetermined using real-time RT-PCR. In experiment6, to explore the role ofTLR4/MyD88signaling in regulating hepatic PI3K/Ak signaling, ICR and MyD88-/-mice were i.p. injected with LPS (1.0mg/kg). Liver sample was collected at2,6and12h after LPS. Phosphorylated Akt, MyD88, P85and PTEN were analyzed by Westernblotting. ICR mice were i.p. injected with LPS (1.0mg/kg). Liver sample was collected2h after LPS. The interaction between MyD88and P85was determined using Co-IP. Inexperiment7, to investigate LPS whether affects insulin-mediated PI3K/Akt signaling,ICR mice were randomly divided into four groups (Control, LPS group, Insulin alonegroup, and Insulin+LPS group). In Insulin alone and Insulin+LPS groups, mice weretreated with insulin (2.0U/kg)6h after LPS. Liver sample was collected5min afterinsulin. Hepatic phosphorylated Akt was analyzed using Western Blotting. Inexperiment8, in order to explore the effects of LPS on hepatic lipid accumulation,C3H/HeJ, C3H/HeN and ICR mice were i.p. injected with LPS (1.0mg/kg) and liversample was collected24h after LPS. Hepatic lipid accumulation was determined by oilred O staining. In experiment9, to explore the effects of LPS on hepatic SREBP-1cactivation, C3H/HeJ, C3H/HeN and ICR mice were i.p. injected with LPS (1.0mg/kg).Liver sample was collected2,6and12h after LPS. Nuclear SREBP-1c was determined using Western Blotting. In experiment10, in order to explore the role of PI3K/Aktsignaling in LPS-induced hepatic SREBP-1c activation, ICR mice were randomlydivided into four groups (Control, LY group, LPS group, and LPS+LY group). In LYand LPS+LY groups, mice were treated with LY294002(50mg/kg, i.p.)30min beforeLPS. Liver sample was collected12h after LPS. Phosphorylated Akt and nuclearSREBP-1c were measured using Western Blotting.Results The results of this study were as follows:(1) LPS caused prolonged reduction ofblood glucose in C3H/HeN and ICR mice but not in C3H/HeJ mice.(2) LPSsignificantly down-regulated mRNA expression of hepatic g-6-pase and pepck.(3) GTTresults showed that glucose tolerance decreased in LPS-treated C3H/HeN and ICR micebut not in C3H/HeJ mice. ITT results showed that insulin tolerance increased inLPS-treated C3H/HeN and ICR mice but not in C3H/HeJ mice.(4) The level of hepaticAkt phosphorylation was significantly increased in LPS-treated C3H/HeN and ICRmice but not in C3H/HeJ mice.(5) LPS didn’t affect the mRNA expression of irs-1andirs-2, in liver. Insulin caused significant increases in the phosphorylation of IRS-1tyrosine residues. Although LPS alone had no effect on the phosphorylation of IRS-1tyrosine residues, it slightly suppressed insulin-activated phosphorylation of hepaticIRS-1tyrosine residues.(6) LPS significantly upregulated hepatic MyD88expression.Moreover, LPS stimulated the interaction between MyD88and P85in liver. In addition,LPS had no effect on hepatic Akt phosphorylation in MyD88-/-mice.(7) Either LPS orinsulin alone induces hepatic Akt phosphorylation. Interestingly, an additive effect wasobserved in hepatic Akt phosphorylation when LPS and insulin were injected.(8) LPSinduced hepatic lipid accumulation in LPS-treated C3H/HeN and ICR mice but not inC3H/HeJ mice.(9) LPS remarkably increased nuclear SREBP-1c in liver of C3H/HeNand ICR mice but not C3H/HeJ mice.(10) PI3K inhibitor significantly inhibitedLPS-induced hepatic Akt phosphorylation and blocked hepatic SREBP-1c activation. Conclusion The present results allow us to reach the following conclusions. First,TLR4/MyD88signaling is involved in the regulation of hepatic glucose and lipidmetabolism; second, TLR4/MyD88-mediated regulation of hepatic glucose and lipidmetabolism is associated with the interaction between MyD88and P85, the regulatorysubunit of PI3K signaling, and subsequent Akt phosphorylation; third, Aktphosphorylation contributes, at least partially, to LPS-induced activation of hepaticSREBP-1c, which is a key transcription factor in the regulation of liver fatty acid andtriglyceride synthesis; and finally, this process may lead to the increased synthesis ofhepatic fatty acid and TG and lipid accumulation.