Shepherd Emma L, Karim Sumera, Newsome Philip N, Lalor Patricia F
Centre for Liver and Gastroenterology Research, Birmingham National Institute for Health Research, Birmingham Biomedical Research Centre, Institute of Immunology and Immunotherapy, University of Birmingham, Birmingham B15 2TT, West Midlands, United Kingdom.
World J Hepatol. 2020 Nov 27;12(11):931-948. doi: 10.4254/wjh.v12.i11.931.
Non-alcoholic fatty liver disease (NAFLD) is associated with obesity, insulin resistance and dyslipidaemia and currently is estimated to affect up to a third of all individuals in developed countries. Current standard of care for patients varies according to disease stage, but includes lifestyle interventions common insulin sensitizers, antioxidants and lipid modifiers. However, to date specific therapies have shown little histological or fibrosis stage improvement in large clinical trials, and there is still no licensed therapy for NAFLD. Given the high prevalence, limited treatment options and significant screening costs for the general population, new treatments are urgently required.
To assess the potential for inhibition of the amine oxidase enzyme vascular adhesion protein-1 (VAP-1) to modify hepatic lipid accumulation in NAFLD.
We have used immunochemical and qPCR analysis to document expression of VAP-1 and key functional proteins and transporters across the NAFLD spectrum. We then utilised hepatocytes in culture and human precision cut liver slices in concert with selective enzyme activity inhibitors to test the effects of activating the semicarbazide-sensitive amine oxidase activity of VAP-1 on hepatic lipid uptake and triglyceride export. A murine model of NAFLD was also used to determine the consequences of VAP-1 knockout and gene expression arrays were used to quantify the effects of VAP-1 activity on key lipid modifying and proinflammatory gene expression.
We confirmed that increasing severity of NAFLD and progression to cirrhosis was associated with a significant increase in hepatocellular VAP-1 expression. Hepatocytes exposed to recombinant VAP-1 and its substrate methylamine showed increased lipid accumulation as determined by quantification of Oil Red O uptake. This was recapitulated using hydrogen peroxide, and lipid accumulation was accompanied by changes in expression of the lipid transporter molecules FABP3, FATP6, insulin receptor subunits and PPARα. Human liver tissue exposed to recombinant VAP-1 or substrates for endo/exogenous VAP-1 produced less triglyceride than untreated tissue and demonstrated an increase in steatosis. This response could be inhibited by using bromoethylamine to inhibit the SSAO activity of VAP-1, and mice deficient in VAP-1/AOC3 also demonstrated reduced steatosis on high fat diet. Exposure of human liver tissue to methylamine to activate VAP-1 resulted in increased expression of FABP2 and 4, FATP3-5, caveolin-1, VLDLR, PPARGC1 and genes associated with the inflammatory response.
Our data confirm that the elevations in hepatic VAP-1 expression reported in nonalcoholic steatohepatitis can contribute to steatosis, metabolic disturbance and inflammation. This suggests that targeting the semicarbazide sensitive amine oxidase capacity of VAP-1 may represent a useful adjunct to other therapeutic strategies in NAFLD.
非酒精性脂肪性肝病（NAFLD）与肥胖、胰岛素抵抗和血脂异常相关，目前据估计在发达国家影响高达三分之一的人群。目前针对患者的标准治疗方案因疾病阶段而异，但包括生活方式干预、常用的胰岛素增敏剂、抗氧化剂和脂质调节剂。然而，迄今为止，在大型临床试验中，特定疗法在组织学或纤维化阶段改善方面收效甚微，且尚无获批用于NAFLD的疗法。鉴于其高患病率、有限的治疗选择以及普通人群的高额筛查成本，迫切需要新的治疗方法。
评估抑制胺氧化酶血管黏附蛋白-1（VAP-1）对改善NAFLD肝脏脂质蓄积的潜力。
我们使用免疫化学和定量聚合酶链反应（qPCR）分析来记录VAP-1以及NAFLD范围内关键功能蛋白和转运蛋白的表达。然后，我们将培养的肝细胞和人精密肝切片与选择性酶活性抑制剂协同使用，以测试激活VAP-1的氨基脲敏感胺氧化酶活性对肝脏脂质摄取和甘油三酯输出的影响。还使用NAFLD小鼠模型来确定VAP-1基因敲除的后果，并使用基因表达阵列来量化VAP-1活性对关键脂质修饰和促炎基因表达的影响。
我们证实，NAFLD严重程度增加及进展为肝硬化与肝细胞VAP-1表达显著增加相关。通过定量油红O摄取测定，暴露于重组VAP-1及其底物甲胺的肝细胞显示脂质蓄积增加。用过氧化氢重复此实验，脂质蓄积伴随着脂质转运分子脂肪酸结合蛋白3（FABP3）、脂肪酸转运蛋白6（FATP6）、胰岛素受体亚基和过氧化物酶体增殖物激活受体α（PPARα）表达的变化。暴露于重组VAP-1或内源性/外源性VAP-1底物的人肝组织产生的甘油三酯比未处理组织少，并显示脂肪变性增加。使用溴乙胺抑制VAP-1的SSAO活性可抑制这种反应，VAP-1/AOC3基因缺陷的小鼠在高脂饮食时也显示脂肪变性减少。将人肝组织暴露于甲胺以激活VAP-1导致FABP2和4、FATP3 - 5、小窝蛋白-1、极低密度脂蛋白受体（VLDLR）、过氧化物酶体增殖物激活受体γ辅激活因子1（PPARGC1）以及与炎症反应相关基因的表达增加。
我们的数据证实，非酒精性脂肪性肝炎中报道的肝脏VAP-1表达升高可导致脂肪变性、代谢紊乱和炎症。这表明靶向VAP-1的氨基脲敏感胺氧化酶活性可能是NAFLD其他治疗策略的有用辅助手段。
