肝脏的代谢分区氧气水平对代谢的影响 Metabolic zonation of the liver: The oxygen gradient revisited

　

肝脏的代谢分区氧气水平对代谢的影响

Metabolic zonation of the liver: The oxygen gradient revisited

　

摘要：

肝脏具有维持全身动态平衡所必需的多种功能。这要求各种代谢途径能够以最有效的方式并行运行，并且无用的循环保持最小。在很大程度上，这是由于被称为代谢分区（metabolic zonation)的肝实质的功能特化而实现的，其通常在肝脏疾病中丢失。

虽然这种现象已知大约40年，但潜在的监管途径尚未完全阐明。生理上发生的氧气梯度被认为对分区的出现至关重要;然而，在过去十年中的许多报告表明β-连环蛋白信号传导和刺猬蛋白（Hh）途径促成代谢分区可能已经改变了这种观点。在目前的综述中，我们将这些新的观察结果与肝脏腺泡内的氧梯度作为分区调节器的概念联系起来。许多事实表明β-连环蛋白和Hh途径可以通过缺氧信号系统和缺氧诱导转录因子（HIF）调节，这强调了这一点。总而言之，我们提供了一种观点，通过这种观点，所有这些途径之间的动态相互作用可以驱动肝脏分区，从而有助于其生理功能。

1.简介

肝脏是负责维持血糖水平，氨代谢，异生素和代谢的内源代谢副产物的生物转化和以及胆汁合成的中心代谢器官。所有这些过程都需要以最有效的方式并行运行许多途径和酶反应。为实现这一目标，肝实质显示出一种称为代谢分区的功能组织。虽然多年来一直被称为特定现象，但仍然很大程度上不知道哪种监管机制可以建立这种模式。在过去十年中的研究表明，除了来自血流的动态因子之外，Wnt / b-连环蛋白途径在确定区带模式中起主要作用。从后者可以看出，氧和活性氧（ROS）能够促成代谢分区的某些特征。目前的审查旨在将新的观察结果与可以推动肝脏分区的调节过程及其对肝脏疾病的潜在影响之间的理解联系起来。

2.解剖学，功能单元和分区

乍一看，肝脏的宏观和微观解剖结构给出了非常均匀的组织成分印象。然而，就细胞类型和功能组织而言，肝脏是异质的（综述参见[1]）。在组织学水平上，小叶代表最小的单位[2]。传统上它几乎呈六角形，中间有一条中央静脉。六边形的角由所谓的门静脉三联体形成，其由来自门静脉的分支（也称为末端门静脉）和来自肝动脉的分支（也称为末端肝小动脉以及胆管）组成（图1A）。在小叶中，肝脏的实质细胞肝细胞彼此连接并且作为绳索可见。绳索从中央静脉向门脉三联体辐射出来。在肝脏细胞膜中，肝细胞膜相互连接，并在任一侧面对称为血窦的血液通道。窦状隙几乎可以被认为是用有孔内皮细胞系包裹的管;窦状隙也含有常驻巨噬细胞，库普弗细胞。重要的是，内皮细胞衬里和肝细胞顶膜之间有一个小空间，叫做Space of Disse;它参与淋巴引流并为储存脂肪和维生素A的星状细胞提供了一个居住点[2]（图1B）。

　

　

图1.肝脏微观结构，氧气梯度和代谢分区。（A）经典六角形肝小叶，中央有中央静脉（CV），门静脉三联征（PT）角，门静脉分支也称为门静脉末端（TPV，蓝点），肝动脉分支也称为末端肝动脉（THA，红点）以及胆管（BD，绿点）。腺泡从PT延伸到两个相邻的中央静脉的方向。可以区分三个区域。 1，门脉带; 2，中介区; 3，perivenous，pericentral或centrilobular区域。（B）肝窦和氧梯度。肝细胞（HC）彼此连接，胆小管（BC）将HC中形成的胆汁输送到胆管（BD）中。用有孔的内皮细胞（EC）包裹正弦曲线。 HC和EC由Disse空间隔开，该空间是肝星状细胞（HSC）的居住位置。常驻巨噬细胞，Kupffer细胞（KC）也可以在正弦曲线中找到。（C）主要代谢途径的分布。 pp，门槛; pv，perivenous; AA，氨基酸; Cho，胆固醇合成; CYP，细胞色素P450酶; Ggn，糖原;乳酸，乳酸; GPX，谷胱甘肽过氧化物酶; GS，谷氨酰胺合成; GST，谷胱甘肽转移酶。（有关此图例中对颜色的引用的解释，读者可参考本文的Web版本。）

　

　

虽然小叶(Lobule)代表一个更结构性的单位，但肝脏腺泡（Liver acinus)被认为是血流量的功能单位[2]。通过将两个门静脉三联体与一条线连接起来，可以看到腺泡，该线延伸到两个相邻的中央静脉的方向。最初区分两个区域，一个围绕门静脉三联体，即（门静脉区域），第二个区域围绕中央静脉（周围，周围或中心小叶区域）。与此同时，还考虑了另一个中间区域（区域2）（审查见[1]）（图1A）。尽管在组织学水平上几乎同质的外观，肝小叶/腺泡在亚细胞[3]，[4]，生化和生理功能方面显示出巨大的异质性[5]。因此，发现各种途径的关键酶以及因此代谢能力优先在一个或另一个区域中（图1C），这种模式通常被称为代谢分区[6]。分区为主要器官和全身动态平衡提供了几个优势。例如，它允许相对的通路在空间上分开，这防止了对共同基板和无用循环的竞争。此外，可以连接互补途径，并且可以在具有最佳底物提供的位点进行底物需求活性。虽然并非所有代谢活动都需要进行分区，但已经显示代谢分区的碳水化合物，氨基酸，脂质，氨和异生物质代谢（在许多优秀评论中都有所涉及[7]，[8]，[9]，[10]， [11]，[12]，[13]（图1C））。此外，非实质细胞的定位和功能活动也是带状的[1]，[14]。

　

分区是相当动态的而不是静态的，因为大多数基因表达模式和因此酶分布响应于营养，药物，激素和其他血液传播因子而改变。虽然谷氨酰胺合成酶长期以来被认为是静态分区的一个例子，但最近的研究结果显示其表达区域可以响应甲状腺激素[15]，[16]和rspondins（RSPO）[17]而改变这种观点。因此，现在也可以认为它是动态的，尽管改变其表达的信号可能是稀缺的。

3.涉及分区调节的因素

多年来，许多研究结果产生了不同的概念，可以用这些概念解释分区模式。所有这些，例如流式肝[18]，发育[19]，细胞基质[20]和后分化模式[21]概念都有其优缺点。到目前为止，后分化模式概念似乎提供了一个非常全面的观点。在该概念中，诸如Wnt，刺猬蛋白，激素或诸如HGF的生长因子等形态发生物的梯度以及诸如氧的其他因素共同起作用，以便将基因表达限制于位于肝脏腺泡的特定区域中的分化的肝细胞。因此，它们似乎以分层方式起作用，其中形态发生素的梯度是碱性的，营养物，激素，肝内形成的前列腺素，细胞因子和氧的梯度是调节性的，具有非常显着的影响。

腺泡内的血流和肝脏代谢，而不是自主神经系统和生物基质的差异，似乎对改变梯度的产生至关重要[22]，[23]。来自门静脉分支和门静脉中的肝动脉的血液作为混合物通过血窦流动到中央静脉。由于新陈代谢和消除，血液的组成变化和底物，产物，激素和氧的梯度形成。后者特别重要，范围从门静脉血液中约60-65 mm Hg（84-91μmol/ L）到周围血液中约30-35 mm Hg（42-49μmol/ L）[24]。因此，细胞内pO2低约15mmHg，即门静脉细胞中45-50mmHg和周围细胞中15-20mmHg（综述参见[22]，[23]）（图1B）。这符合线粒体数量和结构的差异[3]，[4]，[25]以及门静脉和周围区域的氧化能力[26]，[27]。

由于氧气构成活性氧（ROS）形成的基础，因此也存在腺泡内氧化还原梯度的可能性。实际上，从灌注的肝脏获得的氧化还原图像显示出从门静脉到周围区域的S形梯度曲线[28]，[29]。

氧气作为碳水化合物代谢调节剂的重要性早已为灌注大鼠肝脏[30]，[31]和培养的原代肝细胞[32]，[33]，[34]的研究所知。虽然氧气对氨基酸，氨和脂质代谢的作用研究较少，但最近的数学模型指出了氧在脂质代谢中的作用，并强调了供氧不足对脂肪肝形成的作用[13]，[ 35]。

氧气作为分区调节剂的作用的体内证据来自于观察到在人类红细胞生成素（EPO）基因转基因小鼠的肝脏中，仅在较少需氧的周围肝细胞中检测到人类EPO mRNA [36]。

4.缺氧诱导型转录因子（HIFs）

关键酶的区带分布主要受到带状基因表达模式的控制，该模式在很大程度上是差异转录因子作用的结果。介导响应氧可用性变化的基因表达调节的重要转录因子是缺氧诱导因子（HIF）。迄今已知三种HIF转录因子（HIF-1α，HIF-2α和HIF-3α）（综述见[37]）。它们在异二聚体复合物中起作用，其中β亚基由ARNT蛋白代表。异二聚体与靶基因中称为缺氧反应元件（HRE）的DNA区域结合，从而增加或减少它们的转录[38]。 HIF-1α和HIF-2α具有重叠但也有不同的靶基因（综述参见[37]），并且有人提出HIF-1α是对低pO2的急性反应的原因，而HIF-2α对更久的慢性缺氧状态有反应[39]。关于HIF-3α的知识要少得多;已经检测到几种剪接变体，它们甚至被认为具有抑制功能[40]，[41]，[42]。

与肝脏中的氧气梯度一致，在较少需氧的周围区域发现所有HIFαs水平较高[43]。在具有肝细胞特异性HIF-1α缺陷的小鼠的研究中举例说明了HIF转录因子对肝脏结构维持的重要性。这些小鼠表现出肝小叶的延长，增加的小叶耗氧量和mtDNA含量的增加[44]。此外，HIF调节主要的代谢肝功能，特别是其中ARNT或两个HIFα亚基缺失的研究支持这一点。小鼠中常见的βHIF亚基（ARNT）的肝特异性消融增加了喂养的胰岛素水平，糖原异生，脂肪生成和酮体减少[45]。 HIF-1α的缺失显示肝脏再生过程中糖原异生[46]，而HIF-2α对肝脏胰岛素信号传导至关重要[47]，因此其在小鼠肝脏中的组成型活化导致与脂肪酸β受损相关的严重肝脂肪变性的发展。 beta- 氧化，降低脂肪生成基因表达，增加脂质储存能力[48]。

响应于氧气的HIF的丰富度主要受蛋白质降解水平的调节。在常氧条件下，HIFα亚基在特定的脯氨酸残基处被含有脯氨酰羟化酶结构域的蛋白质（PHD，由egln基因编码）羟基化;其中四个是已知的[49]。羟基化HIF然后由von Hippel-Lindau（VHL）蛋白结合，该蛋白作为多蛋白泛素连接酶复合物的E3底物识别组分，其泛素化HIF并因此标记它们用于蛋白酶体降解[50]，[51]。

在缺氧条件下，PHD活性降低;因此HIF稳定，转运到细胞核，在那里它们与它们的β-亚基一起与靶基因的HRE结合。延长的缺氧期，通过HIF-1依赖性反馈循环激活PHD2和PHD3表达，导致至少HIF-1α的再羟化和降解[52]。根据HIF依赖性PHD诱导，肝脏的发现显示PHD显示出在中央静脉周围较少需氧区域具有更强表达的带状分布模式[53]。

关于HIF的转录激活，值得注意的是提到另一种氧敏感的羟化酶，称为抑制因子的HIF（FIH）[54]。 FIH充当天冬酰胺羟化酶，其在常氧条件下羟基化HIF-1α和HIF-2α的C末端反式激活结构域中的关键天冬酰胺残基，从而防止共激活因子CBP / p300的结合[21]，[55]。重要的是，PHD对于氧气具有高Km（Km~230-250μM-~21％O2），而FIH的Km约低90μM（~8％）[56]，[57]。这对它们对HIF的作用具有影响，其效果是可以以双相方式调节HIF的N末端反式激活结构域和C末端反式激活结构域的活性[58]。通过这种方式，中度缺氧如90μM（~8％）会对PHD活性产生强烈影响，但不会对FIH产生影响，而更严重的缺氧例如~1％会抑制PHD和FIH;然而，到目前为止还没有为FIH描述过分区模式。

尽管关于PHD和HIF存在许多共同方面，但PHD功能对HIF同种型的影响也存在更具体的差异。例如，已显示肝脏特异性PHD2缺失诱导HIF-1α，而PHD3缺失主要促进HIF-2α诱导，这与小鼠的发现一致，其中小鼠中PHD1，PHD2和PHD3的同时遗传失活重新激活肝脏的表达。 HIF-2靶基因促红细胞生成素（EPO）和刺激红细胞合成[59]。在代谢方面，发现PHD2抑制可改善葡萄糖和脂质代谢，并可预防肥胖和代谢功能障碍[60]。

除了氧之外，已经显示许多信号物质，应激诱导物和信号传导途径能够在常氧下，即在氧和ROS的存在下调节HIF。感兴趣的读者可以更详细地参考一些涵盖该主题的评论[61]，[62]，[63]，[64]，[65]，[66]。简而言之，重金属，激素如胰岛素，生长和凝血因子如HGF，IGF，凝血酶，细胞因子如TNFα，脂多糖（LPS）和机械应力已被证明通过涉及一个或多个细胞内信号通路如磷脂酰肌醇-3-激酶（PI3K）蛋白激酶B / Akt，mTOR，p70S6K1和RAS / RAF / ERK1 / 2以及线粒体代谢物如PHD / FIH底物α-酮戊二酸,有助于这种调节[65] 。

　

5. HIF，PHD，氧化还原和分区

已经显示ROS在许多上述途径中是重要的信号传导分子，并且它们在HIF信号传导中也起重要作用（综述参见[66]）。 HIF-1α和HIF-2α均可通过ROS以直接和间接方式修饰。直接调节需要氧化还原因子-1（Ref- [1]）的存在，并影响Cys800上HIF-1α和Cys848上HIF-2α的反式激活[67]以及共激活因子如类固醇受体辅激活因子-1和转录的募集。中介因素2 [68]。另一种直接的氧化还原作用是HIF-2α的DNA结合结构域中存在的Cys的氧化，而不是HIF-1α的氧化[69]。 ROS的间接作用是通过调节PHD，FIH，氧化还原敏感性激酶和磷酸酶来介导的（综述参见[66]，[70]）。如上所述，PHD在关键的脯氨酸残基处羟基化HIF-1α和HIF-2α，从而在常氧条件下诱导HIF降解。 PHD属于氧，Fe2 +，2-氧代戊二酸和抗坏血酸依赖性双加氧酶家族，在每个催化循环后需要自由基循环系统来再生铁[71]。尽管抗坏血酸是铁再生的关键因素，谷胱甘肽可以替代缺乏维生素C合成的小鼠，指出巯基氧化/还原循环的重要性[72]。同时，其中一个PHD中的一对半胱氨酸残基被描述为调节其氧化还原敏感性，再次突出了硫醇氧化调节PHD活性的潜力，尽管在缺氧的内皮细胞中，没有观察到PHD半胱氨酸氧化的变化[ 73。

HIF-1α和HIF-2α均可被NOX4 [74]或线粒体复合物III的Qo位点产生的ROS降解[75]，[76]。从形成的ROS开始，过氧化氢似乎对HIF调节具有重要意义，因为过量表达谷胱甘肽过氧化物酶或过氧化氢酶，而不是过氧化物歧化酶1或2，阻止了HIF-1α的低氧稳定[75]，[76]，[77] ]。 ROS一起构成了HIF调节的重要环节，特别是与线粒体活性和丰度的改变有关[78]。在这方面，重要的是要注意HIF-1通过从COX4-1到COX4-2调节亚基的细胞色素c氧化酶中的亚基开关减少低氧下的ROS产生，同时提高复合物IV的效率[79]。 HIF-1还诱导丙酮酸脱氢酶激酶1和4，具有阻断丙酮酸进入线粒体的作用[80]。此外，HIF-1通过BNIP3 [81]和miRNA-210 [82]的表达促进缺氧条件下的线粒体自噬[82]，阻断氧化磷酸化所需的Fe / S簇的组装，从而防止ROS形成和细胞死亡增加。关于HIF和线粒体代谢调节的所有这些发现都非常符合线粒体数量和结构以及氧化能力的区域差异[3]，[25]，[26]，[27]和perivenous区域。

此外，最近发现肝脏中的自噬受PI3K-蛋白激酶B / Akt-FOXO3谷氨酰胺合成酶表达网络调控也与上述发现一致[83]。 FOXO3谷氨酰胺合成酶依赖性自噬主要发生在周围区[84]，其中FOXO3似乎主要表达和活跃。这个特征很好地发现了FoxO3a转录可以被MEF和NIH3T3成纤维细胞中的HIF-1α上调[85]和小鼠肾小球微血管内皮细胞系中的PHD抑制剂二甲基肟甘氨酸上调[86]。此外，PHD1（egln2）对FOXO3的脯氨酰羟基化通过抑制其与遍在蛋白特异性蛋白酶家族成员之一（USP9x）的相互作用使其不稳定[87]。

如上所述，已知HIFα信号传导与PI3K / Akt和RAS / RAF / ERK1 / 2级联反应，其中ROS作为激活剂[88]，[89]，[90]。此外，p38 MAPKs和p38上游激酶MKK3和MKK6 [91]显示参与凝血酶[92]和铬（VI）诱导HIF-1α[91]。此外，这些物质还可以在几种细胞类型中诱导HIF-1αmRNA水平[93]，[94]，[95]，[96]。通过比较肝肿瘤的门静脉和肝周围肝细胞的表达模式与活化的Ha-RAS突变，提出生长因子信号，例如通过RAS / RAF / ERK1 / 2途径起作用的HGF对肝脏分区很重要[97] 。尽管HGF在肝脏再生过程中主要作为肝细胞的有丝分裂原[98]，[99]，[100]在周围区域比活动区具有更高的活性[101]，但已知HGF能够刺激HIF活性[ 102]通过氧化还原敏感的转录因子NFκB[94]。

类似的，HIF-1α是NFκB的直接靶基因[103]，[104]，[105]，[106]，[107]，也被发现可被缺氧诱导[108]，并且在对各种刺激，例如乙醇反应中产生ROS [109]。因此，缺氧和ROS也调节NFκB依赖性HIF-1α转录[104]，[107]，这可能对分区产生影响。有趣的是，NFκB的表达和NFκB的核分布显示出区带模式。在大鼠肝脏中，整个NFκBp65亚基表达在门静脉区域的肝细胞中更高。然而，更重要的是，NF-κBp65在周围区域的肝细胞中显示出主要的核定位[110]，与同一区域中较高HIFαmRNA表达的发现一致[43]。

调节HIF系统的另一种氧化还原调节的转录因子是Nrf2（Nfe212;核因子（红细胞衍生的2）样2）[111]。已知Nrf2有助于中间和异生物质代谢，胆汁生成，以及肝再生和癌发生[112]，[113]，[114]，[115]，[116]，[117]，[118]。缺乏Nrf2的肝脏大小减少，三分之二的Nrf2破坏的小鼠显示出血管化受损，先天性肝内分流的出现将门静脉直接连接到下腔静脉。这种先天性肝内分流减少了小叶中心缺氧并降低了周围Cyp2e1的表达，而磷酸烯醇丙酮酸羧激酶，通常局限于门静脉区域，表现出门静脉和周围表达模式[119]。

此外，适当的血管发育和肝窦的形态发生显示依赖于血管内皮生长因子（VEGF）[120]，另一种缺氧诱导基因[121]。因此，细胞间串扰也很重要，并且在发育过程中肝脏上皮细胞系缺乏VEGF会破坏带状内皮细胞和肝细胞分化，以及形成三维血管和区带结构[120]。

在体内，这些信号传导途径本身不单独起作用，因此很容易设想HIF受到互连信号传导的调节，这可能随着时间和在某些生理或病理条件下诱导或延长HIFα丰度。例如，由于血流急剧减少引起的缺血稳定了HIF-1α[122]。此外，再灌注期间产生的ROS可以使PHD失活，还导致HIFα积累。因此，以下重塑过程再次改变氧气和营养供应，这也对HIF水平和信号传导有影响。因此，肝脏的较少需氧的周围区域（即~4-8％O2）组织pO2的微小变化或非缺氧途径的激活可能对肝功能具有高度显着性。

　

　

　

Abstract

The liver has a multitude of functions which are necessary to maintain whole body homeostasis. This requires that various metabolic pathways can run in parallel in the most efficient manner and that futile cycles are kept to a minimum. To a large extent this is achieved due to a functional specialization of the liver parenchyma known as metabolic zonation which is often lost in liver diseases. Although this phenomenon is known for about 40 years, the underlying regulatory pathways are not yet fully elucidated. The physiologically occurring oxygen gradient was considered to be crucial for the appearance of zonation; however, a number of reports during the last decade indicating that β-catenin signaling, and the hedgehog (Hh) pathway contribute to metabolic zonation may have shifted this view. In the current review we connect these new observations with the concept that the oxygen gradient within the liver acinus is a regulator of zonation. This is underlined by a number of facts showing that the β-catenin and the Hh pathway can be modulated by the hypoxia signaling system and the hypoxia-inducible transcription factors (HIFs). Altogether, we provide a view by which the dynamic interplay between all these pathways can drive liver zonation and thus contribute to its physiological function.

　

Keywords

ROS, Antioxidants, Antioxidative enzymes, Matrix, Diet, Fibrosis, HomeostasisHypoxia, HIF, Liver Metabolic zonation Metabolism Morphogen signaling Optimization Pathology Regulatory network Sinusoid Hepatocytes

1. Introduction

The liver is the central metabolic organ responsible for maintaining blood glucose levels, ammonia metabolism, for biotransformation of xenobiotics and endogenous metabolic byproducts of metabolism, as well as for bile synthesis. All these processes require that a number of pathways and enzyme reactions are running in parallel in the most efficient manner. To achieve this, the liver parenchyma displays a functional organization known as metabolic zonation. Although known as a specific phenomenon for years, it is still largely unknown which regulatory mechanism(s) establish this pattern. Research during the last decade has shown that the Wnt/b-catenin pathway plays a major role for determining the zonal pattern in addition to the dynamic factors coming from the blood flow. From the latter, oxygen and reactive oxygen species (ROS) have been shown to be able to contribute to some features of metabolic zonation. The current review aims to connect the new observations and the improved understanding between the regulatory processes that can drive liver zonation and their potential impact on liver diseases.

2. Anatomy, functional units and zonation

At a first glance the macro and micro anatomy of the liver gives a quite uniform impression of tissue composition. However, the liver is heterogeneous in terms of cell types and functional organization (for review see [1]). On the histological level the lobule represents the smallest unit [2]. Classically it is of almost hexagonal shape with a central vein in the middle. The corners of the hexagon are formed by so called portal triads consisting of a branch from the portal vein also called terminal portal vein, and a branch from the hepatic artery also called terminal hepatic arteriole as well as a bile duct (Fig. 1A). In the lobule the parenchymal cells of the liver, the hepatocytes, are connected with each other and are visible as cords. The cords radiate out from the central vein towards the portal triads. In the cords the hepatocyte membranes are interconnected and face blood channels called sinusoids at either side. The sinusoids can almost be considered as tubes wrapped with lines of fenestrated endothelial cells; sinusoids are also populated with resident macrophages, the Kupffer cells. Importantly, there is a small space between the endothelial cell lining and the apical membrane of the hepatocytes called the Space of Disse; it is involved in lymph draining and provides a residence niche for Stellate cells which store fat and vitamin A [2] (Fig. 1B).

　

Fig. 1. Liver micro architecture, the oxygen gradient and zonation of metabolism. (A) Classic hexagonal shaped liver lobule with a central vein (CV) in the middle and portal triad (PT) corners with branch from the portal vein also called terminal portal vein (TPV, blue dot), and a branch from the hepatic artery also called terminal hepatic arteriole (THA, red dot) as well as a bile duct (BD, green dot). The acinus extends from a PT into the direction of two adjacent central veins. Three zones can be distinguished. 1, the periportal zone; 2, the intermediary zone; 3, the perivenous, pericentral, or centrilobular zone. (B) Liver sinusoid and oxygen gradient. Hepatocytes (HC), are connected with each other, bile canaliculi (BC) transport the bile formed in HC into the bile duct (BD). Sinusoids are wrapped with fenestrated endothelial cells (EC). HC and EC are separated by the Space of Disse which is the residence niche for hepatic stellate cells (HC). Resident macrophages, the Kupffer cells (KC) are also to be found in the sinusoid. (C) Distribution of major metabolic pathways. pp, periportal; pv, perivenous; AA, amino acids; Cho, cholesterol synthesis; CYP, cytochrome P450 enzymes; Ggn, glycogen; Lac, lactate; GPX, glutathione peroxidase; GS, glutamin synthesis; GST, glutathione transferase. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

While the lobule represents a more structural unit, the hepatic acinus is considered to be the functional unit in terms of blood flow [2]. The acinus can be visualized by connecting two portal triads with a line from which it extends into the direction of the two adjacent central veins. Initially two zones, one around the portal triads, i.e. (the periportal zone), and a second one around the central vein (the perivenous, pericentral, or centrilobular zone) were distinguished. In the meantime, an additional intermediary zone (zone 2) is also considered (for review see [1]) (Fig. 1A). Despite the almost homogenous appearance on the histological level, the liver lobules/acini display an enormous heterogeneity with respect to subcellular [3], [4], biochemical and physiological functions [5]. Accordingly, the key enzymes of various pathways and thus the metabolic capacities are found to be preferentially in one or the other zone (Fig. 1C), a pattern which became commonly known as metabolic zonation [6]. The zonation provides several advantages to main organ and whole body homeostasis. For example, it allows that opposing pathways are spatially separated which prevents competition for a common substrate and futile cycles. Further, complementing pathways can be linked, and substrate demanding activities can be carried out at sites with the best substrate provison. Although not all metabolic activities need to be zonated, metabolic zonation has been shown for carbohydrate, amino acid, lipid, ammonia, and xenobiotic metabolisms (covered in many excellent reviews [7], [8], [9], [10], [11], [12], [13] (Fig. 1C)). Moreover, the localization and functional activities of nonparenchymal cells also are zonated [1], [14].

Zonation is rather dynamic and not static since most gene expression patterns and consequently enzyme distributions change in response to nutrition, drugs, hormones, and other blood borne factors. Although glutamine synthetase was long considered to be an example of static zonation, recent findings showing that its expression area can extend in response to thyroid hormones [15], [16] and rspondins (RSPO) [17] changed this view. Accordingly, it can now be also considered to be dynamic, though the signals changing its expression may be scarce.

3. Factors involved in the regulation of zonation

Over the years a number of findings gave rise to different concepts with which the pattern of zonation could be explained. All of them, such as the streaming liver [18], developmental [19], the cell–matrix [20], and the post-differentiation patterning [21] concept have their pros and cons. Up to now, it appears that the post-differentiation pattern concept provides a quite comprehensive view. In this concept, gradients of morphogens such as Wnt, hedgehog, hormones or growth factors such as HGF, and other factors such as oxygen act in concert, in order to restrict gene expression to differentiated hepatocytes located in specific zones of the liver acinus. Thereby, they appear to act in a hierarchical fashion with the gradients of morphogens being basic and the gradients of nutrients, hormones, intrahepatically formed prostanoids, cytokines, and oxygen being modulatory with quite significant impact.

The blood flow and liver metabolism within the acinus, rather than differences in the autonomic nervous system, and biomatrix, appear to be crucial for the generation of the modifier gradients [22], [23]. The blood coming from the branches of the portal vein and the hepatic artery in the portal tracts flows as a mixture through the sinusoids to the central vein. Due to metabolism and elimination the composition of blood changes and gradients of substrates, products, hormones, and oxygen are formed. The latter is of particular importance and ranges from about 60–65 mm Hg (84–91 µmol/L) in the periportal blood to about 30–35 mm Hg (42–49 µmol/L) in the perivenous blood [24]. Accordingly, the intracellular pO2 is about 15 mm Hg lower, i.e. 45–50 mm Hg in periportal cells and 15–20 mm Hg in perivenous cells (for review see [22], [23]) (Fig. 1B). This goes in line with differences in the number and structure of mitochondria [3], [4], [25] as well oxidative capacities in periportal and perivenous zones [26], [27].

Since oxygen constitutes the basis for formation of reactive oxygen species (ROS), it is plausible that also an intra-acinar redox gradient exists. Indeed, redox images obtained from perfused livers showed a gradient curve that decreased sigmoidally from the periportal to the pericentral region [28], [29].

The importance of oxygen as a regulator of carbohydrate metabolism has long been known from studies in perfused rat livers [30], [31] and cultured primary hepatocytes [32], [33], [34]. While the role of oxygen for amino acid, ammonia, and lipid metabolism has been less studied, a recent mathematical model points to a role of oxygen in lipid metabolism and stresses the role of insufficient oxygen supply for the development of steatosis [13], [35].

In vivo evidence for the role of oxygen as a modulator of zonation came from the observation that in livers of mice transgenic for the human erythrpoietin (EPO) gene, human EPO mRNA was detected only in the less aerobic perivenous hepatocytes [36].

4. Hypoxia-inducible transcription factors (HIFs)

The zonal distribution of key enzymes is mainly controlled by a zonated gene expression pattern which is to a large extent the result of differential transcription factor action. Important transcription factors mediating the regulation of gene expression in response to changes in the oxygen availability are hypoxia-inducible factors (HIFs). To date three HIF transcription factors (HIF-1α, HIF-2α, and HIF-3α) are known (for review see [37]). They act in a heterodimeric complex where the beta subunit is represented by an ARNT protein. The heterodimer binds to DNA areas known as hypoxia responsive elements (HREs) in target genes thereby increasing or decreasing their transcription [38]. HIF-1α and HIF-2α have overlapping but also distinct target genes (for review see [37]) and it has been suggested that HIF-1α is responsible for an acute response to low pO2, whereas HIF-2α responds to states with more chronic hypoxia [39]. Much less is known about HIF-3; several splice variants have been detected which are even supposed to have an inhibitory function [40], [41], [42].

In line with the oxygen gradient in liver, all HIFαs were found with higher levels in the less aerobic perivenous zone [43]. The importance of HIF transcription factors for structural maintenance of the liver is exemplified in studies from mice with hepatocyte-specific HIF-1α deficiency. Those mice displayed an extension of hepatic lobules, an enhanced lobular oxygen consumption and an increased content of mtDNA [44]. Further, HIFs regulate major metabolic liver functions, and in particular studies in which either ARNT or the two HIFα subunits were deleted supported this. Liver-specific ablation of the common beta HIF subunit (ARNT) in mice increased fed insulin levels, gluconeogenesis, lipogenesis, and decreased ketone bodies [45]. Deletion of HIF-1α was shown to impair gluconeogenesis during liver regeneration [46] whereas HIF-2α was crucial for hepatic insulin signaling [47] and accordingly its constitutive activation in mouse liver resulted in development of severe hepatic steatosis associated with impaired fatty acid beta-oxidation, decreased lipogenic gene expression, and increased lipid storage capacity [48].

The abundance of HIFs in response to oxygen is primarily regulated on the level of protein degradation. Under normoxia HIFα subunits become hydroxylated at specific proline residues by prolyl hydroxylase domain-containing proteins (PHDs, encoded by the egln genes); four of them are known [49]. Hydroxylated HIFs are then bound by the von Hippel-Lindau (VHL) protein which serves as an E3 substrate recognition component of a multiprotein ubiquitin ligase complex that ubiquitylates HIFs and thus marks them for proteasomal degradation [50], [51].

Under hypoxia, PHD activity is reduced; hence HIFs are stabilized, transported to the nucleus where they, together with their beta-subunits, bind to HREs of target genes. Extended periods of hypoxia, activate PHD2 and PHD3 expression via a HIF-1-dependent feedback cycle that leads to a rehydroxylation and degradation of at least HIF-1α [52]. In line with the HIF-dependent PHD induction are findings from liver showing that the PHDs showed a zonal distribution pattern with stronger expression in the less aerobic areas around central veins [53].

With respect to transcriptional activation of HIFs, it is noteworthy to mention another oxygen-sensitive hydroxylase, called factor inhibiting HIF (FIH) [54]. FIH acts as an asparagin hydroxylase which under normoxia hydroxylates a critical asparagine residue in the C-terminal transactivation domain of HIF-1α and HIF-2α, and thereby prevents binding of the coactivator CBP/p300 [21], [55]. Importantly, PHDs have a high Km (Km ~230–250 μM – ~21% O2) for oxygen, whereas the Km of FIH is with about 90 μM (∼8%) lower [56], [57]. This has impact for their action on HIFs with the effect that the activity of the activity of the N- terminal transactivation domain and the C-terminal transactivation domain of HIFs can be regulated in a biphasic manner [58]. In this way, moderate hypoxia such as 90 μM (∼8%) would lead to strong effect on PHD activity but not on FIH, whereas more severe hypoxia such as ~1% would inhibit both, PHDs and FIH; however, no zonation pattern has been so far described for FIH.

Despite the fact that there are a number of common aspects with respect to PHDs and HIFs, there are also more specific differences in the effect of PHD function with respect to the HIF isoforms. For example, liver specific PHD2 deletion has been shown to induce HIF-1α, whereas PHD3 deletion promoted primarily HIF-2α induction which is in line with findings from mice where simultaneous genetic inactivation of PHD1, PHD2, and PHD3 in mice reactivates hepatic expression of the HIF-2 target gene erythropoietin and stimulates red blood cell synthesis [59]. With respect to metabolism it was found that PHD2 inhibition improves glucose and lipid metabolism as well as protects against obesity and metabolic dysfunction [60].

Apart from oxygen, a number of signaling substances, stress inducers and signaling pathways have been shown to be able to regulate HIFs under normoxia, i.e. in the presence of oxygen and ROS; the interested reader is referred to a number of reviews covering that topic in more detail [61], [62], [63], [64], [65], [66]. In brief, heavy metals, hormones such as insulin, growth and coagulation factors such as HGF, IGF, thrombin, cytokines such as TNFα, lipopolysaccharides (LPS), and mechanical stress have been shown to contribute to that regulation by involving one or more intracellular signaling pathways such as the phosphatidylinositol-3-kinase (PI3K) protein kinase B/Akt, the mTOR, p70S6K1, and RAS/RAF/ERK1/2 as well as mitochondrial metabolites such as the PHD/FIH substrate α-ketoglutarate [65].

5. HIF, PHDs, redox and zonation

ROS have been shown to be important signaling molecules in a number of the above mentioned pathways and they also play an important role in HIF signaling (for review see [66]). Both HIF-1α and HIF-2α can be modified by ROS in a direct and indirect manner. Direct regulation requires presence of redox factor-1 (Ref-[1]) and affects transactivation of HIF-1α at Cys800 and of HIF-2α at Cys848 [67] as well as recruitment of coactivators such as steroid receptor coactivator-1 and transcription intermediary factor 2 [68]. Another direct redox effect is oxidation of the Cys present in the DNA-binding domain of HIF-2α, but not HIF-1α [69]. The indirect effects of ROS are mediated via regulation of PHDs, FIH, redox-sensitive kinases, and phosphatases (for review see [66], [70]). As mentioned the PHDs hydroxylate HIF-1α and HIF-2α at critical proline residues thereby inducing HIF degradation under normoxia. The PHDs belong to a family of oxygen, Fe2+, 2-oxoglutarate, and ascorbate dependent dioxygenases which need a radical cycling system to regenerate the iron after each catalytic cycle [71]. Even though ascorbate is a key agent in the regeneration of iron, glutathione could substitute it in mice deficient in vitamin C synthesis, pointing to the importance of thiol oxidation/reduction cycles [72]. In line, a pair of cysteine residues in one of the PHDs was described to modulate its redox sensitivity, again highlighting the potential of thiol oxidation in regulating PHD activity, though in endothelial cells subjected to hypoxia, no variation in PHD cysteine oxidation was observed [73].

Both, HIF-1α and HIF-2α could be prevented from degradation by ROS that were generated by NOX4 [74] or at the Qo site of mitochondrial complex III [75], [76]. From the ROS formed, hydrogen peroxide seems to be of major importance for HIF regulation since overexpression of glutathione peroxidase or catalase, but not superoxide dismutase 1 or 2, prevented the hypoxic stabilization of HIF-1α [75], [76], [77]. Together, ROS appear to constitute an important link for HIF regulation especially in connection with altered mitochondrial activity and abundance [78]. In this respect it is important to note that HIF-1 reduces ROS production under hypoxia via a subunit switch in cytochrome c oxidase from the COX4-1 to COX4-2 regulatory subunit that at the same time increases efficiency of complex IV [79]. HIF-1 also induces pyruvate dehydrogenase kinase 1 and 4 with the effect of blocking pyruvate entry into mitochondria [80]. Moreover, HIF-1 contributes to mitochondrial autophagy under hypoxia via expression of BNIP3 [81] and miRNA-210 [82], which blocks assembly of Fe/S clusters that are required for oxidative phosphorylation and therefore prevent increased ROS formation and cell death. All these mentioned findings with respect to HIF and regulation of mitochondrial metabolism are very much in line with the zonal differences in the number and structure of mitochondria as well oxidative capacities [3], [25], [26], [27] in periportal and perivenous zones, respectively.

Moreover, the recent findings that autophagy in liver is regulated by a PI3K-protein kinase B/Akt-FOXO3 glutamine synthetase expression network are also in agreement with the above mentioned findings [83]. The FOXO3 glutamine-synthetase-dependent autophagy occurs primarily in the perivenous zone [84] in which FOXO3 appeared to be primarily expressed and active. This feature goes nicely along with findings showing that FoxO3a transcription can be upregulated by HIF-1α in MEFs and NIH3T3 fibroblasts [85] and by the PHD inhibitor dimethyloxalyl glycine in a mouse glomerular microvascular endothelial cell line [86]. Moreover, prolylhydroxylation of FOXO3 by PHD1 (egln2) destabilized it by inhibiting its interaction with one of the ubiquitin-specific protease family members (USP9x) [87].

As mentioned above, HIFα signaling is known to undergo a crosstalk with both PI3K/Akt and RAS/RAF/ERK1/2 cascades where ROS act as activators [88], [89], [90]. Further, p38 MAPKs and the p38 upstream kinases MKK3 and MKK6 [91] were shown to be involved in the induction of HIF-1α by thrombin [92] and chromium (VI) [91]. In addition, these substances can also induce HIF-1α mRNA levels in several cell types [93], [94], [95], [96]. By comparing the expression patterns of periportal and perivenous hepatocytes of liver tumors with activating Ha-RAS mutations it was proposed that growth factor signals such as that of HGF acting via the RAS/RAF/ERK1/2 pathway are important for liver zonation [97]. Although HGF acts primarily as mitogen for hepatocytes during liver regeneration [98], [99], [100] with higher activity in the periportal zone than in the perivenous zone [101], it is known that HGF is able to stimulate HIF activity [102] via the redox-sensitive transcription factor NFκB [94].

In line, HIF-1α is a direct target gene of NFκB [103], [104], [105], [106], [107], which was also found to be inducible by hypoxia [108] and ROS generated in response to various stimuli like e.g. ethanol [109]. Thus, hypoxia and ROS regulate also NFκB–dependent HIF-1α transcription [104], [107], that could have an impact on zonation. Interestingly, NFκB expression and nuclear distribution of NFκB displayed a zonal pattern. In rat liver the overall NFκB p65 subunit expression was higher in hepatocytes of the periportal area. However, and more importantly, NF-κB p65 displayed a predominant nuclear localization in hepatocytes of the perivenous area [110], in line with the findings of a higher HIFα mRNA expression in the same area [43].

Another redox regulated transcription factor which regulates the HIF system is Nrf2 (Nfe2l2; nuclear factor (erythroid-derived 2)-like 2) [111]. Nrf2 is known to contribute to intermediary and xenobiotic metabolism, bile production, as well as liver regeneration, and carcinogenesis [112], [113], [114], [115], [116], [117], [118]. Livers deficient in Nrf2 are reduced in size and two thirds of Nrf2-disrupted mice display an impaired vascularization with the appearance of a congenital intrahepatic shunt that directly connects the portal vein to the inferior vena cava. This congenital intrahepatic shunt reduced centrilobular hypoxia and decreased perivenous Cyp2e1 expression while phosphoenolpyruvate carboxykinase, normally confined to the periportal zone, exhibited both a periportal and perivenous expression pattern [119].

Further, proper vascular development and morphogenesis of hepatic sinusoids was shown to be dependent on vascular endothelial growth factor (VEGF) [120], another hypoxia-inducible gene [121]. Thereby, an intercellular crosstalk is also important and lack of VEGF from liver epithelial lineages during midgestational development disturbed zonal endothelial and hepatocyte cell differentiation as well as formation of a three-dimensional vascular and zonal architecture [120].

In vivo, these signaling pathways do not act per se alone and thus it is easy to envision that HIFs are regulated by interconnected signaling which may, with time, and under certain physiological or pathological conditions, induce or prolong HIFα abundance. For example, ischemia due to an acute reduction in blood flow stabilizes HIF-1α [122]. Further, ROS generated during reperfusion can inactivate the PHDs, leading also to HIFα accumulation. Consequently, the following remodeling processes again alter oxygen and nutrient supply which also have an impact on HIF levels and signaling. Thus, it is likely that small changes in tissue pO2 or activation of non-hypoxic pathways in the less aerobic perivenous zone of the liver (i.e. ∼4–8% O2), becomes highly significant for liver function.

6. Beta-catenin and zonation

Interesting seminal discoveries have shown that the Wnt/β-catenin pathway is an important driver of hepatic zonation [123], [124]. Similar to HIFs, β-catenin abundance is regulated post-translationally and in the absence of Wnt signals, β-catenin resides in a multiprotein complex together with glycogen synthase kinase-3 (GSK3), adenoma polyposis coli (APC), CK1, Axin, and the protein Dishevelled (DVL). Within this destruction complex, β-catenin becomes phosphorylated by GSK3 which is the prerequisite for recruitment of the ubiquitin ligase β-TRCP that ubiquitylates β-catenin; thus, marking it for degradation by the proteasome [125], [126]. In the canonical Wnt pathway, binding of Wnt ligands to its receptor, Frizzled, and co-receptors, such as the low-density lipoprotein receptor-related protein 5 (LRP5) and LRP6 [127], [128], destroys the destruction complex. As a consequence, β-catenin becomes stabilized and is transported into the nucleus where it acts as a coactivator for the transcription of Wnt target genes by binding to transcription factors from the T-cell factor (TCF) and lymphoid enhancer factor (Lef) family [129].

Stabilized and active β-catenin is found in perivenous hepatocytes, whereas the negative regulator of β-catenin, APC, is predominantely localized in periportal hepatocytes [123]. Importantly, genetic ablation of APC activates the β-catenin pathway also in the periportal zone. Reciprocally, inhibition of β-catenin signaling in the liver acinus leads to a periportal phenotype in the perivenous hepatocytes [123].

Experiments with fetal rodent hepatocyte cultures supported the role of the Wnt/β-catenin system for metabolic zonation. When these hepatocytes were differentiated in culture to mature hepatocytes only periportal gene expression markers could be detected. However, when the cells were differentiated in the presence of a β-catenin activator this induced a reversible expression of perivenous marker genes [130], [131].

Recent findings have indicated that proteins from the Rspondin (Rspo) family are important Wnt pathway activators [132]. Accordingly, conditional deletion of Rspo3 in mice abrogates proper perivenous zonation. Further, overexpression of another Rspo member, Rspo1, induced expression of perivenous marker genes periportally indicating that Rspo members may be important angiocrine signals modulating the β-catenin-dependent liver zonation [133].

Rspondins possess their own receptors, the LGRs, which together with Rspo prevent clearance of frizzled receptors from the membrane. As a consequence, Wnt signaling is promoted [134]. The LGR system has been shown to be of importance for stem cell renewal, a process also evidently necessary for liver regeneration. In particular LGR5, but not LGR4, is expressed at high levels in damage-activated liver stem cells [135] and with a zonated expression pattern; LGR5 mRNA was exclusively found in perivenous hepatocytes [136], the area showing the lowest oxygen content but an active Wnt/β-catenin signaling in adult livers. In line with these observations are current findings indicating that deletion of LGR4/5 in mouse liver deregulated the expression of periportal and perivenous marker genes such as glutamine synthetase and reduced liver weight [17]. Further, the LGR ligand Rspo3 shows a highly restricted expression pattern in the liver acinus and can be detected only in the endothelial cells of the central vein. Interestingly our own preliminary findings from hepatocytes and HepG2 cells cultured under hypoxia indicated that LGR5 mRNA is induced by hypoxia whereas Rspo´s are not, which indicates that the LGR-β-catenin connection can be controlled by the oxygen gradient in the liver acinus.

7. Hypoxia, HIFs, and β-catenin signaling: a new liaison

Although an interplay between hypoxia, HIFs, and β-catenin signaling was not yet directly shown for liver zonation, there are several reports showing an interrelation between β-catenin and hypoxia signaling. In particular it was found that lower oxygenated adult brain areas exhibit an enhanced Wnt/β-catenin signaling in-vivo. Exposure of mice to chronic hypoxia (10% O2 for 6–72 h) stimulated the activation of Wnt/β-catenin signaling, and activated neurogenic cell proliferation in the subgranular zone of the hippocampal dentate gyrus. Concomitant with exposure to 10% O2, HIF-1α and β-catenin levels were increased and Dvl3 phosphorylation as well as transcription of Wnt target genes in the hippocampus was stimulated [137]. Further, hypoxia increases β-catenin signaling in cultured neonatal hippocampal stem cells [138] and embryonic stem cells (ESCs) [139]. This induction was shown to be mediated by the HIF system since deletion of the hif-1a gene and the HIF-beta encoding gene arnt diminished expression of Wnt/β-catenin target genes including Dkk-4, Lef-1 and Tcf-1 under hypoxia [139]. Further, it was found that upon exposure of cells to hypoxia, HIF-1α can directly bind to the Lef1 and TCF1 gene promoters [139] and in consequence promote the transcriptional activity of β-catenin.

In β-catenin-deficient mouse liver HIF-1α signaling was reduced and affected by the cellular redox balance indicating a role of β-catenin as coactivator of HIF-1α signaling [140]. Indeed, HIF-1α was found to directly interact with β-catenin, thereby competing with TCF-4. DNA–protein interaction analyses revealed that the HIF-1α-β-catenin interaction occurs at HIF-1 target gene promoters [141]. Thus, these results suggest that β-catenin promotes HIF-1-mediated transcription, adaptation to hypoxia, and cell survival.

The expression of perivenous genes was also associated with the interaction of β-catenin with LEF1 and hepatocyte nuclear factor 4-α (HNF4α) that could also be detected with higher levels in the perivenous area [142]. HNF4α is known to act together with HIFs as transcriptional activator for hypoxia-dependent erythropoietin expression [143] which can be detected perivenously during fetal development. Despite the perivenous predominance of HNF4α, it appeared to act as repressor of perivenous β-catenin regulated genes such as glutamine synthetase (GS) and cytochrome P450 2e1 which in the absence of HNF4α appeared in the periportal zone [144]. Interestingly, deficiency of Cited2, another coactivator of HNF4α and also a HIF target gene [145], lead to a disorganized sinusoidal architecture, as well as impaired lipid metabolism and hepatic gluconeogenesis [146] suggesting that hypoxia mediated feedback mechanisms exist which may be controlled at the level of coactivator recruitment.

Further, in response to hypoxia β-catenin was shown to move from the plasma membrane to the cytoplasm where it binds and stabilizes the zink finger containing snail superfamily member SNAI2 and carbonic anhydrase 9 (CA9) mRNAs, in cooperation with the mRNA stabilizing protein HuR, a process which is important for the onset of cancer stem cell features [147]. These findings link hypoxia signaling with the regulation of RNA abundance which is largely affected by the microRNA pathway. Dicer, the key endoribonuclease that processes precursor microRNAs into mature microRNAs was found to be involved in the regulation of zonation; lack of Dicer led to a loss of the periportal pattern and to a diffuse expression of phosphoenolpyruvate carboxykinase, E-cadherin, arginase 1, and carbamoyl phosphate synthetase-1 within the entire acinus [148]. Although this pattern was similar to that seen upon loss of β-catenin, the authors did not find down-regulation of Dicer1 or any microRNAs in β-catenin-deficient liver and suggested involvement of an indirect mechanism. Interestingly, Dicer1 was found to be inducible by hypoxia and to be necessary for the function of HIFs and full expression of HIF target genes [149] from which some are eventually β-catenin repressors.

While the above mentioned findings indicate the links between hypoxia and β-catenin regulation, it is also of utmost importance that the expression of the negative β-catenin regulator APC is suppressed by hypoxia and HIF [150]. Upon exposure of several cell lines to hypoxia (1% O2) APC levels were decreased. This decrease was found to be transcriptionally mediated via HIF-1α since depletion of HIF-1α with siRNA restored the APC levels. Further analyses identified a functional hypoxia-responsive element in the APC promoter. Reciprocally, APC was able to mediate a repression of HIF-1α; a process which, in addition to wildtype APC, required low levels of β-catenin and NFκB activity [151]. Importantly, in this context the action of APC appears to involve several cellular compartments such as the nucleus, and mitochondria [152], [153], [154]. The latter are being found at higher number in the periportal zone and constitute major sites of ROS production [3], [4], [25]. Both, β-catenin and HIF-1α signaling are modulated by ROS [155] and in particular superoxide and H2O2 are considered to serve as messengers in this regulation [65]. Hence, changes in the oxygen availability and consequently in superoxide production may have a major influence on β-catenin and HIF-1α signaling as well as on zonation. Indeed, hepatocyte-specific deletion of manganese superoxide dismutase (MnSOD; sod2) which generates H2O2, caused a disruption of the zonal gene expression [156]. Further, HIF-1α as well as β-catenin were absent in hepatocyte-specific MnSOD-deficient mice which were also more prone to chemically-induced carcinogenesis [157].

Together, these findings are in line with the view that the low oxygen content in perivenous hepatocytes leads to induced HIF function which mediates APC repression and consequently stabilization and nuclear localization of β-catenin. Vice versa, loss of HIF function in the more oxygenated and ROS enriched periportal zone allows full APC expression with the result that β-catenin signaling is suppressed.

8. Hedgehog signaling

Recently it was proposed that Hedgehog (Hh) signaling, that is in particular important during development and regeneration, has also a determining role for metabolic zonation [158]. In adult liver, Hh signaling is low in hepatocytes whereas hepatic stellate cells and cholangiocytes are Hh positive [159], [160]. In particular Hh signaling becomes activated upon certain states of liver damage as seen in non-alcoholic fatty liver disease (NAFLD), liver cirrhosis, and hepatocellular carcinoma (HCC) as well as after partial hepatectomy [161], [162]. The known Hedgehog ligands (Sonic-Hh, Indian-Hh, and Desert-Hh) exert their effects after binding to Patched (PTCH1, -2) receptors on the surface of Hh-responsive cells. In the canonical Hh pathway this relieves the inhibitory actions of PTCH's on the signaling co-receptor Smoothened (SMO) which then promotes the nuclear localization and activation of the glioma-associated oncogene transcription factors GLI1, GLI2, and GLI3 [163].

Mouse liver displayed a perivenous zonation of IHh [164] and conditional hepatocyte-specific deletion of SMO resulted in periportal lipid accumulation and up-regulation of key lipogenic transcription factors such as SREBP and PPARs and enzymes such as FASN [165]. The latter displayed a reversed zonation in the SMO deleted mice; instead of being perivenous [8], [166] like in the control mice, FASN was detected in the periportal zone of the SMO knockouts. Further, from the Gli transcription factors especially Gli3 appeared to be responsible for the observed effects, in particular in the SMO-deletion mediated development of steatosis [165]. Importantly, cholesterol biosynthesis, glycogen content, and glycolysis were not altered in hepatocyte-specific SMO deleted mice suggesting that the major role of the Hh pathway consist in, but is not limited, to regulate lipid metabolism and its zonation.

Interestingly, the regulation of IGF1 and IGFBP1 which were found to be expressed in the periportal and perivenous area, respectively [167], was also affected in the same SMO knockout model; Deletion of SMO decreased IGF1 but increased IGFBP1 expression [168].

9. Hypoxia, HIFs, and hedgehog signaling: another liaison

Although the findings with respect to lipid metabolism as well as IGF1 and IGFBP1 expression in the hepatocyte specific SMO-deficient mice support a role of Hh signaling for metabolic zonation, they correlate also well with the role of oxygen in the regulation of zonation.

With respect to fatty acid synthesis it has been shown that FASN can be induced by hypoxia [169]. Thereby, hypoxic induction of FASN appears to be an indirect HIF effect involving first HIF-dependent up-regulation of SREBP1 and then an action of SREBP1 on the promoter of FASN [169]. By contrast and similar to APC, HIF-1 inhibited β-oxidation by suppressing expression of medium- and long-chain acyl-CoA dehydrogenases (MCAD and LCAD) [170] which is in line with the old findings that under starvation, oxidation of FAs is more pronounced in periportal hepatocytes.

The regulation of IGF1 expression by oxygen is supported from findings showing that serum IGF-1 levels were lower in cyanotic than in acyanotic congenital heart disease patients [171], [172] which fits with the reduced perivenous IGF1 appearance. The role of oxygen, ROS, and the HIF system was much more explored with respect to IGFBP1 expression in primary rat hepatocytes where perivenous oxygen tensions enhanced IGFBP-1 expression. Experiments with the iron chelator desferrioxamine and H2O2 supported the concept that ROS and the HIF system are involved [173]. Interestingly and in line with experiments from zebrafish [174], the induction of IGFBP1 appeared to be mediated by HIF-3α and HIF-2α, and to a lesser extent by HIF-1α. The participation of the HIF system was further supported by experiments targeting the HIF proline hydroxylases [173].

Although these findings suggest that the Hh and HIF pathways may act in a separate manner, ample evidence exists for a connection between the HIF pathways with the Hh signaling.

The interconnection between the Hh and hypoxia response pathway became first evident by findings showing that hypoxia can induce expression of the Hh ligand SHh, the pathway activity marker Patched1, and subsequently a systemic Hh response in adult mice [175]. Interestingly, the Hh response followed the accumulation of HIF-1α and various HIF inhibitory approaches revealed that lack of HIF-1α blunted hypoxia-dependent Hh activation [175]. Further, it was shown that also SMO can be transcriptionally induced in response to hypoxia in various cell models [176], [177]. In addition, hypoxia was also able to act on GLI1 transcription factors without SMO via PI3K or ERK1/2 signaling. Furthermore, the hypoxia effects on Hh signaling were not limited to HIF-1α but also shown to involve HIF-2 α, depending on the cell type [178].

Remarkably and similar to the connection between HIF and β-catenin pathways, there appears to exist also a feed-back regulation between the HIF and the Hh system. In particular, the SHh-GLI1 pathway was able to upregulate HIF-2α levels under normoxic conditions [178]. Predominantly this crosstalk appears to be of importance during liver damage, where cholangiocytes and myofibroblasts secrete Hh ligands in order to promote survival and proliferation of both cell types [159], [160]. Upon liver damage, hepatic stellate cells become activated myofibroblasts via an Epithelial-to-Mesenchymal-Transition (EMT) and the Hh pathway was shown to be a major regulator of the stellate cell to myofibroblast transition [160]. During this transition a metabolic switch occurs which is in favor of aerobic glycolysis and GlI transcription factors and HIF-1α appeared to be involved in this switch. In mice with different types of liver injury genetical or pharmacological inhibition of SMO, attenuated HIF-1α expression and suppressed glycolytic gene expression. Reciprocally, activating SMO up-regulated HIF-1α mRNA expression, and chromatin immunoprecipitation assays (ChIP) identified that GLI proteins interact with the hif-1a promoter [179].

Moreover, the Hh pathway undergoes also a cross talk with the Wnt/β-catenin signaling pathway. This crosstalk appears to be mutual and even complex with the involvement of common components but different functional aspects of the GLI transcription factors and tissue specificity. In adult tongue epithelium dominant activation of β-catenin lead to a significant up-regulation of Shh which then diminished β-catenin signaling [180]. Vice versa, IHh is a target of Wnt/β-catenin, and in liver it has been shown that the perivenous area expressing IHh is extended upon activation of β-catenin signaling [164]. However, these regulations may further divide at the level of GLI or TCF transcription factors. In quail embryos GLI2 and GLI3 were shown to be regulated by Wnt/β-catenin signaling whereas GLI1 activation in somites was shown to be controlled by SHh signaling [181]. In mouse chondrocytes activation of hedgehog signaling selectively inhibited β-catenin-induced FGF18 expression. The selectivity was shown to be due to the Hh mediated induction of a dominant negative isoform of TCF7L2 (dnTCF7L2) in so called interzone progenitor cells [182]. Moreover, Wnt/β-catenin signaling can have repressive effects on SHh signaling in a number of cancer cells [183]. For example SHh signaling was high during differentiation of gastric cancer cells, whereas Wnt signaling was decreased during differentiation [184]. Altogether, it is obvious that an intricate interplay between Wnt and SHh signaling occurs in which oxygen availability has an important role, although the detailed mechanisms remain still largely unclear, hence more investigations are required to further clarify the role of the Wnt/Hh crosstalk for metabolic zonation in liver.

10. Conclusion

The multitude of functions including being the major metabolic organ puts the liver into an important strategic position for maintaining whole body homeostasis. Metabolic zonation of the liver is an important feature that helps to achieve this. Research during the past decade provided considerable information into the complex underlying networks involved in liver zonation. In particular, these studies have established a major role for β-catenin signaling, and the involvement of the Hh pathway in metabolic zonation. These findings are still well in accordance with the “old” concept that the oxygen gradient within the liver acinus is a regulator of zonation. This is underlined by a number of facts showing that β-catenin signaling and the Hh pathway can be modulated by the HIF system. Given the zonation of non-parenchymal cells such as bile duct cells and hepatic stellate cells which are both predominantely found in the portal tract and periportal area, respectively, they may well be secretors of Hh signals. The Hh signals in turn could, by leaving a gradient, spread into the perivenous direction. In the oxygen rich periportal area Hh signaling could, at least in a large part, inhibit β-catenin signaling. The low oxygen content in the perivenous zone would, via the HIF system, activate β-catenin, and LGR5 expression as well as suppress APC expression. These, together with Rspondins secreted from the central vein endothelial cells, may lead to an active β-catenin/TCF genetic program in perivenous hepatocytes. To keep balance and to maintain homeostasis, hypoxia activates expression of Hh components IHh, SMO and likely GLI1 at the same time (Fig. 2).

　

Fig. 2. Impact of the oxygen gradient on β-catenin and hedgehog signaling and metabolic zonation. Non-parenchymal cells such as bile duct cells and hepatic stellate cells are more abundant in the oxygen rich periportal area and secrete Hh signals (nPC Hh) and inhibit β-catenin signaling. The low oxygen content in the perivenous zone activates the HIF system, induces LGR5 expression and activates β-catenin as well as suppresses expression of the negative β-catenin regulator APC. Rspondins secreted from the central vein endothelial cells, activate β-catenin via LGR5 in perivenous hepatocytes. To maintain homeostasis, hypoxia activates expression of Hh components in hepatocytes (HC Hh) to feedback inhibit β-catenin.

The view that the oxygen gradient is an organizing principle for tissue structure and organization by mediating a critical balance between several regulatory pathways such as β-catenin and hedgehog may be of critical importance for physiological and pathological as well as developmental processes in liver and in general. Nonetheless, the picture of liver zonation does not yet have the highest resolution. In order to improve it, more comprehensive mechanistic analyses are necessary. This may include experiments designed to unravel the complex interplay between the oxygen gradient, ROS, and the different liver cell types. At the cellular level this encompasses investigations with respect to the role of long non-coding RNAs, miRNAs, and different epigenetic modifications. Thereby, novel techniques and improved animal models will help to solve conflicting data which may exist due to technical limitations in older studies. Finally, this will lead to a much clearer picture about the role of the oxygen gradient and ROS action in liver, and improve our understanding of several diseases associated with hypoxia and ROS such as ischemia/reperfusion injuries, NAFLD, NASH, and hepatocellular carcinoma.

Acknowledgements

The authors is grateful to all researchers who contributed to the field and apologizes to all those whose work could not be cited due to space limitations. The work was supported by grants from the Academy of Finland (SA 296027), Jane and Aatos Erkko Foundation, Biocenter Oulu, and the European Cooperation in Science and Technology Organization (COST Action BM1203/EU‐ROS).

SOURCE:

Redox Biology

Volume 11, April 2017, Pages 622-630

Faculty of Biochemistry and Molecular Medicine, Biocenter Oulu, University of Oulu, Oulu, Finland

Received 22 December 2016, Revised 12 January 2017, Accepted 13 January 2017, Available online 17 January 2017.

https://doi.org/10.1016/j.redox.2017.01.012Get rights and content

Metabolic zonation of the liver: The oxygen gradient revisited - ScienceDirect https://www.sciencedirect.com/science/article/pii/S2213231716304499

　

　

　