GPR35: mechanisms of action and agonism as a potential therapeutic strategy for non-alcoholic fatty liver diseases

所谓的“肥胖流行病”的后果之一是，近年来与肥胖相关的各种疾病和状况大幅增加。这些疾病包括由肝脏中过量脂肪积累引发的一系列疾病，这些疾病与过度饮酒没有直接联系。这种非酒精性脂肪性肝病(NAFLD)包括一系列状态，从最初肝细胞中脂肪的积聚(脂肪变性)，到炎症(脂肪性肝炎)和肝脏瘢痕形成(纤维化)的额外发展，在极端情况下，可能会导致肝硬变。在英国，NAFLD和非酒精性脂肪性肝炎(NASH)目前影响着大约3%-12%的成年人。尽管这些情况可以通过饮食限制来解决，但目前还没有批准的药物来帮助这一过程，或者当患者发现实现和维持这一过程具有挑战性时可以使用。最近，在研究一种用作人类肝细胞(肝细胞)实验模型的细胞类型时，我们发现临床上用于滴眼液的一种名为洛多沙胺的药物能够防止这些细胞中的脂肪积聚。几年前，在不同的研究中，我们已经证明洛多沙胺能够激活一种名为GPR35的细胞表面受体，但只有当受体是人类版本而不是在老鼠身上发现的版本时才有效。我们继续证明，当我们从肝细胞样细胞中通过基因删除GPR35时，洛多沙胺不再具有这种作用，这表明GPR35肯定参与了这一作用。老鼠经常被用来研究药物和药物的可能影响，然后再决定它们是否适合在人体上进行试验。然而，由于洛多沙胺在小鼠GPR35上不起作用，我们对小鼠进行了改造，使其具有人类形式的这种受体。使用从这些所谓的人源化(转基因)小鼠中提取的肝细胞，我们再次能够证明洛多沙胺在防止脂肪堆积方面非常有效。我们现在计划在这项工作的基础上，试图获得进一步的信心，即在人类体内激活GPR35可能是治疗脂肪肝疾病的有效策略。然而，要提供这样的信心，还有很多工作要做。首先，目前，尽管我们看到了洛多沙胺的有效性，但我们不知道是什么分子机制触发了将GPR35激活与最终有益结果联系起来的机制。我们将在肝细胞模型和人源化小鼠的肝细胞中研究这一点。作为这项工作的一部分，我们将创造另一只带有不同类型人类GPR35受体的转基因小鼠，这将直接帮助我们发现关键机制。此外，人类的GPR35基因比老鼠的GPR35基因更复杂。这导致人类有一种小鼠没有的额外形式的GPR35。因此，我们还将探索这种额外的人类特有的“剪接变异体”的细节，以及它如何对洛多沙胺和其他可以激活GPR35的化合物做出反应。最后，但可能具有最重要意义的是，我们将让这些GPR35转基因小鼠继续服用旨在使它们体重增加并在肝脏积累脂肪的‘高脂肪饮食’，或者修改后的高脂肪饮食，除了促进脂肪积累之外，还会导致类似于人类NASH的肝脏炎症和纤维化。当我们随后在实验结束时分析肝脏、血液和其他组织的病理学时，我们将评估用这种饮食维持的小鼠是否能够限制或防止疾病的发展。这些研究将提供关于GPR35如何在分子水平上发挥作用的基本知识，并将使我们能够评估这些人源化小鼠中GPR35的激活是否提供了足够的证据，开始使用洛多沙胺或其他GPR35激活药物进行可能的临床研究。

One of the consequences of the so called 'obesity epidemic' is that a variety of diseases and conditions linked to it have increased substantially in recent years. These include a spectrum of conditions initiated by the accumulation of excess amounts of fat in the liver that are not linked directly to over-use of alcohol. Such non-alcoholic fatty liver disease (NAFLD) encompasses a range of states from the initial build-up of fat in liver cellss (steatosis), to the additional development of inflammation (steatohepatitis) and liver scarring (fibrosis) that, in extreme cases, can potentially result in liver cirrhosis. NAFLD and non-alcoholic steatohepatitis (NASH) now affect some 3-12% of adults in the U.K. Although these conditions can be addressed by diet restriction there are currently no approved medicines to assist this process or that can be used when patients find this challenging to achieve and maintain. Recently when studying a cell type that is used as an experimental model of human liver cells (hepatocytes) we found that a drug called lodoxamide, that is used clinically in eyedrops, was able to prevent fat accumulation in these cells. Some years ago in different studies we had shown that lodoxamide was able to activate a cell surface receptor called GPR35, but only effectively when the receptor was the human version and not the version found in mice. We went on to show that when we genetically removed GPR35 from the hepatocyte-like cells lodoxamide no longer had this effect, suggesting that GPR35 must be involved. Mice are often used to study possible effects of drugs and medicines before deciding if they are suitable for trials in humans. However, because lodoxamide does not work effectively on mouse GPR35, we have modified mice so they have a human form of this receptor instead. Using liver cells taken from these so called "humanised" (transgenic) mice we were again able to show that lodoxamide was very effective in preventing fat accumulation. We now plan to build on this work to try to gain further confidence that activating GPR35 in humans might be an effective strategy to treat fatty liver diseases. There is, however, much to do to provide such confidence. Firstly, at the moment, although we see how effective lodoxamide is, we do not know what molecular mechanisms are triggered that link GPR35 activation to the final beneficial outcomes. We will study this in both the hepatocyte cell models and in liver cells taken from the humanised mice. As part of this we will create another transgenic mouse with a different type of human GPR35 receptor that will directly help us to discover the key mechanisms. In addition, the gene for GPR35 in humans is more complex than the equivalent one in mice. This results in humans having an additional form of GPR35 that mice do not have. We will therefore also explore the details of this additional human specific "splice variant" and how it responds to lodoxamide and other chemical compounds that can activate GPR35. Finally, but potentially of greatest significance, we will take these GPR35 transgenic mice and maintain them either on a 'high-fat diet' designed to make them gain weight and accumulate fat in the liver or a modified high-fat diet that in addition to promoting fat accumulation also causes liver inflammation and fibrosis similar to human NASH. We will assess whether treating mice maintained on such diets with lodoxamide, is able to limit or prevent disease development when we subsequently analyse the liver, blood and other tissues for pathology at the end of the experiments. These studies will provide both basic knowledge on how GPR35 functions at a molecular level and will allow us to assess if activation of GPR35 in these humanised mice provides sufficient evidence to begin to move towards possible clinical studies using either lodoxamide or other GPR35 activating drugs.