Inhibition Of Hepatic TLR4 Mediated Metaflammation Ameliorates Insulin Resistance Induced By Catch-Up Growth

Abjective:TLR4-mediated hepatic and systemic inflammation as well as hepatic autophagy were observed in catch-up growth induced insulin resistance of C57BL/6j mice. The alterations of above-mentioned parameters were also observed after hepatic TLR4 m RNA expression was selectively suppressed by si RNA packaged by lentivirus.Methods: Forty 6-week-old C57 BL/6j mice were randomly divided as chow diet(n=10) and restrict diet(RD,n=30) group after one week of acclimatization. Chow diet mice were continuously fed a chow diet as normal control(NC). RD mice were reared with 50% caloric of that intaken by their weight-matched NC counterparts for 4 weeks followed by with high fat diet ad libitum for 8 weeks. Then RD mice were randomly divided into three groups, that is catch-up growth(CUG, n=10), catch-up growth with TLR4-p LV intervention(CUGT,n=10)and catch-up growth with Cont-p LV intervention(CUGC, n=10)group and fed on high fat diet for another 4 weeks during which time mice were simultaneously injected with TLR4-p LV or Cont-p LV(1×10^8 Tu/ml, 100 ul per time) once every two weeks via tail vein. The efficiency of TLR4-p LV and expression of hepatic macrophage biomarker genes(F4/80,Cd68,Cd11c) were assessed by RT-q PCR; the activation of IKK?/ NF-?B and JNK/AP-1, two main signaling pathways down macrophages, were measured by ELISA, so as to serum TNF-?and IL-6 concentrations;Proteins(LC3B, AMPK, m TOR) and genes(Beclin1, Atg7) associated with autophagy in liver tissue were detected by western blot and RT-q PCR respectively; hepatic Akt activation was assessed by western blot; blood lipids(FFA, TG, TC) were profiled by biochemical tests; hepatic steatosis was detected by immunohistochemistry; glucose tolerance and systemic insulin sensitivity were observed dynamically.Results: 1. TLR4 m RNA expressions of liver and adipose tissue in CUG group mice were significantly increased compared with NC group(p<0.05), and compared to CUG group, hepatic TLR4 m RNA expressions in mice of CUGT group were significantly decreased(p<0.05), while there was no significantly difference between the CUGT group and NC group in TLR4 m RNA expression of adipose tissue. And TLR4 gene expressions in liver and adipose tissue of CUGC group mice were not significantly different in relative to CUG mice. 2. In liver tissue, general macrophage markers F4/80 and Cd68 m RNA expression were significantly up-regulated in CUG group mice compared to NC mice(p<0.01), and M1 macrophage activated marker Cd11 c gene expression was also increased(p<0.05), the parameters mentioned above in CUGC group changed similarly and there were no statistical difference between CUGC and CUG group. While in CUGT group, m RNA expression of F4/80, Cd68 and Cd11 c were all significantly dropped in relative to CUG group(p<0.05). NF-?B and AP-1 activity in liver of CUG group were increased(p<0.01) which were almost cancelled in CUGT group. Likewise, serum IL-6 and TNF-?concentration were elevated in CUG mice which effect was fully abrogated by TLR4-p LV treatment. 3. CUG mice featured hepatic steatosis according to H&E staining and accompanied by increased hepatic TG content(p<0.01), and compared to this group, hepatic steatosis in CUGC group was almost the same while in CUGT group was improved, so as to hepatic TG accumulation(p<0.01). The activity of Akt in liver was attenuated in CUG mice compared with NC group(p<0.01) while in CUGT group, Akt activation was significantly enhanced compared to CUG group(p<0.05). 4. Compared to NC group, hepatic LC3-II/LC3-I ratios of CUG group mice were decreased(p<0.01), Beclin1 and Atg7 gene expressions were down-regulated, p-AMPK/AMPK ratios dropped to less than 50%(p<0.01) and activation of m TOR were increased(p<0.01); TLR4-p LV intervention could reverse the above deterioration in various degrees while significantly, and kept all the parameters to a level parallel to NC group. 5. Body weight in RD group was continuously dropping during caloric restrict, while rebound promptly to a level a little above NC group with ad libitum access to high fat diet within one week and thereafter kept increasing in a rate parallel to NC group; visceral fat/ weight ratios in CUG mice were increased(p<0.01) while subcutaneous fat/ weight ratios were not significantly affected compared to NC mice, visceral fat/ weight ratio in CUGT group was reduced compared with CUG group(p<0.05) while in CUGC group was not significanlty changed; serum FFA, TG and TC were elevated in CUG group while normalized in CUGT group; glucose tolerance and insulin sensitivity were transiently improved during caloric restriction and deteriorated sharply in the first 4-week high fat diet and progressively developed till the end of experiment, and once again TLR4-p LV could largely normalized glucose tolerance and insulin sensitivity.Conclusion: Catch-up growth mice featured hepatic steatosis, visceral fat accumulation and overt insulin resistance independent of obesity. TLR4-p LV intervention could selectively inhibited hepatic TLR4 gene expression, decreased macrophage infiltration and M1 activation, suppressed IKK ? / NF- ? B and JNK/AP-1 signaling pathways and decreased serum pro-inflammatory cytokines(IL-6 and TNF-?), ultimately improved hepatic and systemic metaflammation. In the other hand, hepatic autophagy improved after TLR4-p LV treatment and it in turn inhibited TLR4-mediated metaflammation or enhanced insulin signaling to ameliorate catch-up growth induced insulin resistance and associated metabolic disorders.