The Roles Of AMPK And MTOR Pathways In The LPS-induced Anorexia

The aim of this study is to understand the interactions between LPS-provokedpro-inflammatory signaling cascades and neuronal regulatory network underlied thedevelopment of anorexia. Two parts are involved in the research. Firstly, screen appetiteregulating genes change by measuring mRNA expression and/or protein phosphorylationlevels caused by LPS in the mice hypothalami; Secondly, confirm the critical signalingpathway by i.c.v. injection of AICAR/or Rapamycin to alleviate the anorexia caused by LPS.Trial18-week-old Kunming mice were housed under a controlled environment of23oCwith12h/12h light/dark (L/D) cycle. Mice were provided with a standard chow and had freeaccess to food and water before the experiment started. All mice were randomly divided intofour groups,6mice in each group, and all mice were housed individually and fasted for24hprior to the experimental day. At the start of dark cycle, mice were injected intraperitoneallyLPS (from Escherichia coli055:B5, Sigma) at dosages of0,10,100,1000μg/kg BW,respectively, and were immediately access to food. Food intake was recorded after1h,2h,4h,6h,8h,12h,18h and24h. Body weight was recorded at time points of0h,24h,48h and72hafter injection. The results showed that food intake was significantly decreased by LPStreatment in a dose-dependent way (p<0.01). Body weight was dosage-dependent. Mice lostmore bodyweight over the first24h, as the doses increased. Thereafter, body weight wasrestored in10and100μg/kg BW groups, rather than the1000μg/kg BW group (p<0.01).The results suggested that anorexia and weight loss was induced by LPS administration in adose-dependent way.Trial28-week-old Kunming mice were housed under a controlled environment of23oCwith12h/12h light/dark (L/D) cycle. Mice were provided with a standard chow and had freeaccess to food and water before the experiment started. All mice were randomly divided intothree groups,12mice in each group, and all mice were individually housed and fasted for24hprior to the experimental day. At the start of dark cycle, mice were injected intraperitoneallyLPS at dosages of0,100,500μg/kg BW, respectively. Two hours after injection, mice weresacrificed and hypothalami were quickly removed. The samples were rapidly frozen in liquid nitrogen and then stored at-80oC for further analysis. The result showed that the mRNAlevels of IL-1α, IL-1β, IL-6and TNF-α were significantly increased compared with control(saline)(p<0.05). Orexigenic appetite genes mRNA expression levels of AgRP and NPYshowed no significant difference compared with control group (p>0.05). In contrast, theexpression of pro-opiomelanocortin (POMC) had a trend to be up-regulated by LPS injectionrelative to control (saline)(p=0.0537). The protein level of phosphorylated NF-кB versus totalNF-кB and β-actin in the hypothalami of the mice was significantly increased after treatmentwith LPS (p<0.05). After LPS administration, the protein content of NF-кB in cell nuclearwas significantly increased compared with in cytoplasm of the mice hypothalami (p<0.05).The phosphorylation levels of FoxO1at Ser256and Thr24/FoxO3a (Thr32) sites were bothshowed a significant increase with LPS treatment. During LPS intraperitoneal injection,protein content of total FoxO1was much more than control in cytoplasm, decreasing proteincontents in nuclear (p<0.05). The phosphorylation level of AMPK at Thr172is significantlydecreased2-hours after LPS administration (p<0.05) and phosphorylation levels of mTORand P70S6K were significantly upregulated by LPS (p<0.05).Trial3Mice were fasted for24h, randomly divided into four groups (n=6per group)marked A, B, C, D then anesthetized with isoflurane before i.c.v. injection of AICAR (12μg/2ul) or vehicle (artificial cerebrospinal fluid,2μl).2h before the start of the dark cycle,group B and D were injected AICAR, the others were received ACSF. One hour later, mice ingroup A and B were given intraperitoneal injection with500μg/kg LPS, other mice wereintraperitoneally injected saline. Cumulative food intake was calculated after1,2,4,6,12and24h. The data showed that pre-treated with AICAR did not attenuate the low food intakeinduced by LPS (p>0.05). The result suggested that anorexia caused by LPS may beindependent of the AMPK pathway in hypothalamus and AMPK pathway might not be thecritical point which involved in LPS-induced anorexia.Trial4Mice were fasted for24h, randomly divided into four groups (n=6per group)marked A, B, C, D then anesthetized with isoflurane before i.c.v. injection of rapamycin (20μg/μl) or vehicle (artificial cerebrospinal fluid,1μl).2h before the start of the dark cycle,group B and D were injected rapamycin, the others were received ACSF.1hour later, micewere intraperitoneally injected with saline or500μg/kg LPS. Thereafter, food intake was recorded after1,2,4,6,12and24h. Two hours after LPS injection, all mice were sacrificedand hypothalami were rapidly removed for further analysis. The results showed thatrapamycin could significantly increased food intake in a dose-dependent manner (p<0.05).After rapamycin treatment, gene expression level of POMC was decreased significantlycompared with the LPS treatment, whereas expression of NPY was significantly increasedrelative to group administrated with LPS (p<0.05). The phosphorylation level of FoxO1atsite of Thr24/FoxO3a (Thr32) was significantly decreased within i.c.v injection of rapamycincompared with LPS treatment group without rapamycin (p<0.05). In the LPS treatment grouppre-treated with rapamycin, the ratios of phosphorylated FoxO1at site of Ser256to totalFoxO1protein had a trend to be down-regulated. Pro-inflammatory cytokines genesexpression levels of TNF-α and IL-1α were significantly reduced and the expression level ofIL-1β had a trend to be downregulated. The mRNA level of IL-6did not vary followingrapamycin treatment. The higher phosphorylated NF-кB in LPS treatment mice had a trend tobe downregualted by rapamycin pre-treatment. The rapamycin administration significantlydecreased the LPS-induced protein phoshorylated level of P70S6K (p<0.05). These resultsindicated that functions of rapamycin might start with decreasing activity of mTOR andreducing production of proinflammatory cytokines, then, contribute to changes of relatedproteins, and ends up with relieving anorexia induced by LPS.