| EFFECTS OF NAMPT GENE DEFICIENCY ONGLUCOSE-LIPID METABOLISM AND INSULINRESISTANCE IN C57BL/6J MICEObjective To investigate the effects of NAMPT deficiency byshRNA-mediated on insulin resistance and glucose-lipid metabolism in highfat diet fed C57BL/6J miceMethods Male C57BL/6J mice were randomly divided into standardchow diet (SCD) fed group (NF group, n=10), high-fat diet fed group (HFgroup, n=10), HFD fed+pAd-shGFP group (GFP group, n=10) and HFD fed+pAd-shNAMPT group (shNAMPT group, n=10). All mice were fed for12weeks. Mice from GFP group and shNAMPT group were injected withAd-GFP or Ad-shNAMPT by tail-vein at the end of11week. Intracariodarterial and intrajugular venous catheters were placed for infusions andblood sampling, respectively. Animals were allowed to recover for3daysfollowing surgery. Then, hyperinsulinemic-euglycemic clamp Studies wereperformed to evaluate insulin resistance and glucose-lipid metabolism inawake mice.Results The mRNA and protein expressions of NAMPT in liver andNAMPT plasma levels in HF, GFP and shNAMPT groups weresignificantly lower than in NF group, respectively, whereas the expressions of NAMPT mRNA and protein and NAMPT plasma levels in shNAMPTgroup were significantly decreased than that in HF and GFP group (bothP<0.01). Compared to mice fed a SCD, body weight, FBG, FIns, TG, TC,FFA, HDL-C,LDL-C,liver TC and TG levels were significantly increased inmice fed a HFD (P <0.01). However, Fins and HDL-C levels in shNAMPTgroup were significantly higher than HF and GFP groups (P<0.05or P<0.01). Hepatic TC content was decreased by Ad-sh NAMPT treatmentcompared with controls (P<0.05).During the steady-state of clamp, plasmaHDL-C was suppressed in all group, but it remained higher in the shNAMPTgroup than in the other three groups (both P<0.01).In NF group, glucoseinfusion rate (GIR) was significantly decreased compared with other threegroups (both P <0.01). Ad-shNAMPT treatment had no effect on GIR inHFD-fed mice. In the end of clamp, glucose disappearance rate (GRd) wassignificantly decreased and hepatic glucose production (HGP) wassignificantly increased in three HFD groups compared with in NF group (allP <0.01). GRdand HGP were also unchanged in the shNAMPT groupcompared with HF and GFP groups (all P>0.05).Conclusion: Insulin resistance models that are similar to T2DM ofhuman beings can be established in C57BL/6J mice induced by HFD.NAMPT deficiency dose not significantly improve insulin resistance inC57BL/6mice. EFFECT OF NAMPT GENE DEFICIENCY ONATHEROSCLEROSIS IN ApoE-/-MICEPARTⅠObjective To investigate the effect of NAMPT gene deficiency on lipidmetabolism and atherosclerosis in ApoE-/-miceMethods Male ApoE-/-mice were randomly divided into high-fat dietfed+PBS group (PBS group, n=10), HFD fed+pAd-shGFP group (GFP-Agroup, n=10) and HFD fed+pAd-shNAMPT group (NAMPT-A group,n=10). All mice were fed for12weeks. At week8and12of HFD feeding,ApoE-/-mice were also treated with Ad-shNAMPT, Ad-GFP (1×109pfu in200μl of PBS) or sterile saline by tail vein. The lipid of plasma and liverwere detected by enzymatic colorimetric methods. The lesion area wasevaluated by hematoxylin and eosin. Meanwhile, we analyzed thecomposition of AS including lipid and collagen respectively by oil red Ostaining and Picrosirius Red and aortic lesions en face by oil red O.Results The mRNA and protein expressions of NAMPT in liver tissuewere decreased and plasma NAMPT levels were reduced in ApoE–/–micetreated with Ad-shNAMPT compared with PBS-and Ad-GFP-treatedcontrol mice (all P<0.01). Treatment of ApoE–/–mice with Ad-shNAMPTraised HDL-C compared with PBS-and Ad-GFP-treated mice (P <0.01).Hepatic TC content was decreased by Ad-sh NAMPT treatment comparedwith controls (P<0.05).The plaque size and lipid deposition of aortic sinuslesions were reduced, respectively in Ad-sh NAMPT-treated micecompared with PBS-and Ad-GFP-treated control mice whereas totallesional collagen content was increased in Ad-sh NAMPT-treated micecompared with that of controls(P<0.01). In addition, the en-face ofdescending aortas of lipid deposition in Ad-sh NAMPT-treated mice wasreduced compared with PBS-and Ad-GFP-treated control mice ((P<0.01). Conclusion NAMPT deficiency may play important role inatherogenesis, and inhibition of NAMPT can reduce plaque size and inducefeatures of plaque stability. PARTⅡObjective To investigate the effects of NAMPT deficiency byshRNA-mediated on macrophage RCT in vivoMethods Male ApoE-/-mice were randomly divided into high-fat dietfed+PBS group (PBS group, n=8), HFD fed+pAd-shGFP group (GFP-Agroup, n=8) and HFD fed+pAd-shNAMPT group (NAMPT-A group, n=8).All mice were fed for12weeks. At week8and12of HFD feeding, ApoE-/-mice were also treated with Ad-shNAMPT, Ad-GFP (1×109pfu in200μl ofPBS) or sterile saline by tail vein.3H-cholesterol-labeled andAcLDL-labeled RAW cells (typically4.5×106cells containing6.4×106counts per minute (cpm) in0.5ml DMEM) were injected intraperitoneallyinto HFD-fed ApoE KO Mice treated with PBS, Ad-GFP or Ad-sh NAMPT.The cholesterol content of the lipid protein fractions was deterimined by fastprotein liquid chromatography (FPLC) analysis. Plasmas, liver and feceswere counted in an LSC. Expression of NAMPT, PPARα, Liver Xreceptor-α (LXRα), ATP binding cassette transporters A-1(ABCA1) andABCG1in the liver were determined by Real-time RT-PCR and western blotanalysis.Results FPLC analysis revealed that the increased plasma cholesterolin Ad-shNAMPT-treated mice was mainly in the HDL region (columnfractions26–31) compared with those in vehicle treated mice.The plasma3H-cholesterol levels in the mice with Ad-shNAMPT treatment weresignificantly higher at6h(6h1.46±0.36vs.1.11±0.22,1.10±0.23),24h(24h5.01±0.83vs.3.5±0.44,3.55±0.62), and48h (48h5.26±0.95vs.3.79±0.75,3.81±0.88) than those in control groups(P<0.05or P<0.01).The liver3H-cholesterol levels in the mice with Ad-shNAMPT were significantlyhigher(6.88±0.79vs.3.88±0.72,3.92±0.73)than those in controlgroups(P<0.05).The feces3H-cholesterol levels in the mice with Ad-shNAMPT were significantly higher(9.85±2.01vs.6.29±1.26,6.31±1.32)than those in control groups(P<0.05). The PPARα, LXRα,ABCA1and ABCG1mRNA and protein contents in the liver were markedlyincreased in Ad-shNAMPT treated mice compared with control mice(P<0.05or P<0.01).Conclusion NAMPT deficiency promoted macrophage RCT inApoE–/–mice. These changes were accompanied by increased PPARα,LXRα, ABCA1and ABCG1expressions in the liver. PARTⅢObjective To investigate the effect of NAMPT deficiency byshRNA-mediated on macrophage cholesterol effluxMethods Cholesterol efflux from3H-cholesterol-labled RAW cellstreated with Ad-GFP or Ad-shNAMPT and incubated with HDL per mlprepared in DMEM-BSA was measured. Hepa1-6cells and RAWmacrophages were transfected with Ad-shNAMPT or Ad-GFP and thenexposed to10μM MK886. Cellular total cholesterol was measured usingcholesterol assay kits. Expression of NAMPT, PPARα, Liver X receptor-α(LXRα), ATP binding cassette transporters A-1(ABCA1) and ABCG1weredetermined by Real-time RT-PCR and western blot analysis.Results In Ad-shNAMPT-treated cells, both NAMPT mRNA andprotein expressions were significantly decreased compared withAd-GFP-treated cells (all P<0.01). Plasma3H-cholesterol levels inAd-shNAMPT-treated macrophages were significantly higher than those inuntreated control cells (P<0.01). Ad-shNAMPT treatment led to a30%and33%decrease respectively in total cholesterol levels compared with vehicletreatment(both P<0.05). However, this effect was negated by thecoadministration of MK886, a selective inhibitor of PPARα (P<0.05).ABCA1, ABCG1, PPARα and LXRα mRNA and protein were significantlyincreased in Ad-shNAMPT treated cells compared with vehicle-treated cells(P<0.05or P<0.01). Importantly, these changes in the mRNA and proteinexpression of genes related to cholesterol metabolism were abolished byMK886treatment (P<0.05or P<0.01).Conclusion NAMPT knockdown facilitated cholesterol efflux inRAW cells and decreased TC content in Hepa1-6and RAW cells throughthe PPARα-LXRα-ABCA1/G1pathway. |
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