外泌体作为代谢中细胞间串扰的介质

金色的田野 2024-04-12 发布于云南

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介绍

外泌体研究是一个快速发展的领域，几乎与生理学和病理生理学的所有领域相交。外泌体是异质的，代表了几乎所有细胞类型释放的更广泛的细胞外囊泡（EV）的组成部分。分泌的外泌体进入间质空间并最终进入循环，成为各种细胞间串扰和通讯系统中的关键成分。外泌体是EV类中的双层囊泡纳米颗粒，大小范围为30至150nm，并通过特定的多步胞吐过程从细胞中释放（Doyle和Wang，2019;Kalluri 和 LeBleu，2020 年;Li 等人，2019c）。微囊泡（MV），有时称为外体，是较大的 EV，从质膜上萌芽，大小为 100 nm-1 μm，凋亡小体是 EV（直径 50-5,000 nm）的更大组成部分，由垂死的凋亡细胞释放（Doyle 和 Wang，2019 年;Kalluri 和 LeBleu，2020 年;Carnino 等人，2020 年）。在这篇综述中，我们将特别关注外泌体、它们的生物发生、货物和生物潜力。

外泌体领域的一个初步概念是，这些纳米颗粒作为一种废物处理系统，允许细胞将不需要的细胞成分包装并分泌到细胞外环境中。虽然这可能仍然部分正确，但最近的研究集中在各种生理和疾病相关事件的外泌体效应子以及作为疾病生物标志物的重要性上。事实上，外泌体的多种信号转导特性现已在各个领域的众多出版物中得到充分认可。在这里，我们将回顾目前对代谢领域外泌体作为疾病生物标志物和代谢调节效应物的理解。由于外泌体以受调节的方式从细胞中分泌，进入循环，并被远端细胞吸收，在那里它们发挥生物学作用，因此外泌体生物学与经典内分泌系统有许多相似之处。事实上，新出现的证据表明，特定货物进入外泌体的负荷及其分泌是可以调节的，从而进一步类比典型的内分泌系统。富集的外泌体群体可以使用基于大小，密度和膜蛋白成分的多种技术从细胞培养基和各种生物体液中分离出来（Dang等人，2020），稍后将讨论。没有单一标准的、公认的外泌体分离技术，但方法正在迅速发展（Théry等人，2018）。尽管外泌体分离方法存在技术局限性、这些颗粒的异质性以及各种表征方法，但外泌体领域已经出现了许多重要的发现。在相当大的程度上，这些努力受到循环外泌体作为生理调节剂和潜在治疗载体以及疾病生物标志物的重要潜力的推动。

作为正常细胞功能的一个组成部分，几乎所有细胞类型都会将各种纳米囊泡释放到细胞外环境中。这些囊泡，通常称为 EV，包含许多货物成分，例如蛋白质、脂质和多种 RNA，包括 mRNA、长链非编码 RNA 和 microRNA （miRNA）（O'Brien 等人，2020 年）。其中一些蛋白质和脂质在表面表达，而另一些则嵌入脂质双层中，这些脂质双层将这些囊泡与囊泡腔内包含的许多其他成分包围在一起。在这些 EV 中，外泌体特别令人感兴趣，因为它们的生物发生和胞外分泌途径包括特定的细胞内机制，可以调节这些机制以确定外泌体组成、功能和靶向。环境和细胞线索，如压力、炎症或细胞周期事件，可以改变加载到外泌体中的特定货物，也可以调节外泌体释放的整个过程（Gurunathan等人，2021）。

使用循环作为运输系统，外泌体到达远端细胞位点，在那里它们可以与细胞表面结合并通过特定机制进行内吞作用。最近的证据表明，外泌体摄取的机制可以被调节，并且外泌体类别可能含有特定的靶向分子，导致对受体组织具有一定程度的特异性（Murphy等人，2019）。外泌体也可以经历转胞吞作用，使它们能够穿过血脑屏障，进入中枢神经系统（Ramirez等人，2018）。国际细胞外囊泡学会（ISEV）建议将EV作为描述具有脂质双层的细胞释放的所有纳米颗粒的通用术语（Théry等人，2018）。外泌体是 EV 的重要组成部分，存在各种技术来分离富集的外泌体群体。然而，外泌体分离技术仍在不断发展，目前的方法产生的制剂在外泌体中高度富集，但仍含有少量的其他 EV 亚型，例如 MV。使用可靠的外泌体制备方法，对所得囊泡进行充分表征，如果承认适当的警告，则使用术语外泌体来描述这些制剂是合理的。在表征方面，ISEV 建议测量至少三种外泌体特征的蛋白质标志物，包括至少一种跨膜蛋白，例如四跨膜蛋白（CD63、CD81 和 CD82）、整合素等。此外，需要一种胞质蛋白（例如，TSG101，ALIX和syntenin）和一个阴性蛋白标记物（例如，白蛋白和核糖体蛋白）（Théry等人，2018）。除了这些标准之外，还在白细胞（CD37和CD53），内皮细胞（EC）（PECAM1），间充质干细胞（MSC）（CD90）和其他细胞中鉴定出细胞类型特异性外泌体蛋白（Théry等人，2018）。在本综述的其余部分，将在适当的时候使用术语外泌体。

外泌体生物发生

外泌体来源于内体结构，内体结构起源于质膜内陷内体的内吞作用（Jella等人，2018;图 1）。这些早期分选内体最终成熟为晚期分选内体，然后可以产生多泡内体结构。从这些后一种颗粒中，多泡体（MVB）通过内体膜的内陷而发育，在这些MVB内形成许多气泡，通常称为腔内囊泡（ILV）（Jella等人，2018）。这些含有ILV的MVB可以与包含降解途径的溶酶体融合。或者，它们可以经历特定的胞吐过程，最终与质膜融合，将内陷的外泌体释放到细胞外空间（Zhang等人，2019）。已经描述了参与外泌体生物发生过程的多种蛋白质和脂质。例如，Rab蛋白调节囊泡交通和外泌体形成（Blanc和Vidal，2018）。此外，其他蛋白质组装成转运所需的内体分选复合物的四个组分（ESCRT-0，-I，-II和-III）以及辅助蛋白，如ALIX等（Colombo等人，2013;Vietri 等人，2020 年）。这些复合物参与MVB和ILV的生物发生。ESCRT-0 复合物识别并隔离内体膜中的泛素化蛋白质，而 ESCRT-I 和 -II 复合物负责将膜变形为具有螯合货物的芽。ESCRT-III组分随后驱动囊泡剪裂（Colombo等人，2013）。四跨膜蛋白是跨膜蛋白，可诱导膜弯曲结构，从而形成囊泡（Andreu和Yáñez-Mó，2014）。最后，各种脂质修饰酶（如鞘磷脂酶）产生促进囊泡形成的神经酰胺（Wortzel等人，2019）。然而，要阐明这些细胞内成分在外泌体生物发生/胞吐过程中的具体作用，仍有许多工作要做。一个复杂的因素是，参与这些过程的许多蛋白质也具有其他细胞内功能，因此功能获得或丧失研究可能同时扰乱外泌体生物发生/胞吐作用和其他细胞内事件。外泌体生物发生的基本过程仍有待完全定义，读者可以参考有关该主题的几篇优秀评论（Bebelman等人，2018;Zhang等人，2019）。

受体细胞对外泌体的摄取

外泌体到达受体细胞后，通过两步过程进行内化。最初，外泌体通过蛋白质 - 蛋白质或受体 - 配体相互作用与细胞表面结合，这可以启动激活内吞途径的信号级联反应（Horibe等人，2018）。

通过膜结合受体摄取

外泌体表面蛋白的作用已通过用蛋白酶K处理外泌体来证明，该外泌体显示受体细胞的摄取减少（Inder等人，2014;Smyth等人，2014）。许多蛋白质已被确定为膜结合受体，能够将外泌体与细胞结合。这可以包括整合素 - 四跨膜蛋白复合物（Hazawa等人，2014;Jankovičová等人，2020），和/或外泌体肝配蛋白受体（Eph）与受体细胞上表达的膜肝配蛋白的结合（Irie等人，2009;Gong 等人，2016 年;Sato 等人，2019 年）。另一个例子是具有胶原结构的巨噬细胞受体（MARCO），它可以介导巨噬细胞的外泌体内化（Kanno等人，2020）。此外，外泌体膜蛋白还调节循环动力学。通过发现在静脉给药前对外泌体进行蛋白酶处理导致清除延迟来证明这一点。由于外泌体表面蛋白受亲本细胞蛋白质库的影响，因此外泌体膜组成可以根据细胞生理状态而变化。外泌体富含糖蛋白，并且由于受体介导的外泌体摄取可能依赖于聚糖（Williams等人，2019），天然糖基化的破坏会改变受体细胞类型的摄取（Williams等人，2019）。糖基化是否直接参与细胞识别和/或主动进入细胞尚不清楚。

内吞作用

内吞作用是网格蛋白依赖性或非依赖性途径摄取外泌体的最常见机制（Mulcahy等人，2014）。网格蛋白介导的内吞作用是细胞外物质摄取的一个众所周知的过程，抑制该途径可减少外泌体的摄取（Murphy等人，2019;Mulcahy等人，2014）。网格蛋白依赖性内吞作用主要涉及小窝蛋白 1、RhoA 和 ARF6（Sandvig 等人，2008 年）。小窝可以介导质膜内特定微结构域的形成，也称为脂筏，它们是外泌体的入口点（Murphy等人，2019）。

巨胞饮作用、吞噬作用和融合

巨细胞增多症涉及在依赖于肌动蛋白聚合的过程中将大量细胞外液摄取到巨松果体中（Lin等人，2020）。在巨胞饮作用期间，由肌动蛋白丝驱动的质膜突起形成内陷，非特异性内吞细胞外液和小颗粒（Lin等人，2020）。已经报道了许多通过该途径摄取外泌体的例子（Fitzner等人，2011;Svensson等人，2013;Tian等人，2014）。

巨噬细胞，树突状细胞和其他细胞类型也通过吞噬作用吸收外泌体（Mulcahy等人，2014）。吞噬作用可能代表免疫细胞清除外泌体的选择性过程（Feng等人，2010;McKelvey 等人，2015 年）。

融合是外泌体内化的另一种潜在机制。在细胞表面膜融合过程中，两种不同的脂质双层膜靠得很近并形成半融合柄。该茎膨胀，出现半融合隔膜双层，随后是熔融孔开口，促进两个疏水核心的混合（Mulcahy等人，2014）。一般过程与靶向机制的潜在作用，指导特定的外泌体亚型摄取到选定的受体细胞中是一个重要且新兴的话题，也是未来研究的关键课题。

电动汽车包含各种各样的货物

电动汽车的封装货物受到保护，不会降解，并在不同的生物流体中保持相对稳定。这些货物包括蛋白质、脂质、代谢物、氨基酸、各种 RNA 种类和 DNA（Veziroglu 和 Mias，2020 年;Mohan 等人，2020 年;Yáñez-Mó 等人，2015 年）。

外泌体货物

外泌体货物赋予外泌体对邻近或远端细胞的生物学作用。外泌体的脂质组成包括鞘脂，胆固醇，磷脂酰丝氨酸，饱和脂肪酸和神经酰胺（Trajkovic等人，2008;Skotland等人，2020），所有这些都可以在质膜中找到。使用减少总体外泌体分泌的中性鞘磷脂酶抑制剂，显示了神经酰胺在ILV萌芽到MVB腔内的直接作用（Menck等人，2017）。外泌体的蛋白质组包括膜运输相关蛋白，例如四跨膜蛋白（CD63、CD81、CD82 和 CD9），它们通过 ALIX 和 ESCRT-III 依赖性途径募集到外泌体（Larios 等人，2020 年）。外泌体也可以富集热休克蛋白（Hsp60，Hsp70和Hsp90），整合素和MHC II类蛋白（Clayton等人，2005）。外泌体不仅仅代表亲本细胞的蛋白质组成。某些蛋白质通过翻译后修饰控制的选择性蛋白质货物分选富集在外泌体中（Carnino等人，2020）。其中之一是与ESCRT复合物结合所需的货物蛋白的泛素化（Carnino等人，2020）。

在不同的外泌体RNA物种中，miRNA和mRNA的研究最充分（Carnino等人，2020）。Valadi等人已经证明，一些外泌体mRNA是完整的，并且可以翻译为受体细胞中的功能蛋白（Valadi等人，2007）。RNA物种被分类到外泌体的过程可能是选择性的，这些机制最近已经过综述（Wei等人，2021;O'Brien 等人，2020 年）。

外泌体DNA（exoDNA）可以包括单链或双链DNA形式的核DNA和线粒体DNA。ExoDNA可以位于囊泡表面或囊泡内部。DNA包装成外泌体的机制尚不清楚（Wortzel等人，2019）。

MV货物

MV还包含上述货物，并且有几种机制调节货物蛋白募集到MV腔中，包括ESCRT（Yáñez-Mó等人，2015）。此外，细胞质蛋白与 MV 管腔产生位点的质膜结合。这种结合基于翻译后修饰，例如肉豆蔻酰化和棕榈酰化，这些蛋白质集中在MV出芽的质膜结构域（Meldolesi，2018）。由于 MV 覆盖显着且异质的尺寸范围（50 nM–1 μM）（Konoshenko 等人，2018 年），因此评估 MV 囊泡大小和货物内容物之间的差异将很有趣。

MV中存在不同的RNA物种，包括mRNA，非编码RNA等（Turchinovich等人，2019）。miRNA（包括引导链和乘客链）和pre-miRNA都可以包装成MV以传递信息（Shu等人，2019）。将miRNA包装到MV中的确切机制尚不清楚，但MV与其亲本细胞之间miRNA含量的差异表明miRNA选择性地加载到MV中（Groot和Lee，2020）。MV 中的 DNA 货物包括单链 DNA、双链基因组 DNA 和线粒体 DNA。DNA 的长度各不相同，很大程度上取决于与其相关的 MV 的大小（Elzanowska 等人，2021 年;Bruno 等人，2020 年）。完整的MV DNA可以是>2 Mbp，并且与组蛋白（H 2B）相关，表明存在完整的染色体DNA（Vagner等人，2018）。

miRNA生成、加工、加载和分泌的生物学

miRNA是∼22-nt-长的非编码RNA，是后生动物和植物中基因表达的关键转录后调节因子，在基因表达的转录后调控中起着重要作用（Filipowicz等人，2008）。 miRNA调节大量细胞功能，其表达的变化与几种人类病理生理状态有关。许多miRNA以组织特异性或发育阶段特异性方式表达，有助于mRNA和蛋白质表达的细胞类型特异性谱（Ivey和Srivastava，2015;Kloosterman 和 Plasterk，2006 年）。

miRNA加工

Pri-miRNA 是发夹状的前体 miRNA 分子，由 RNA 聚合酶 II 从独立基因或蛋白质编码基因的内含子转录而成（图 2）。pri-miRNA 可作为 RNase III 家族成员 Drosha 和 Dicer 两种酶的底物。Drosha 裂解导致 ∼70-nt 的前 miRNA 输出到细胞质，其中 Dicer 将其加工成 ∼22-bp miRNA/miRNA 双链体（Michlewski 和 Cáceres，2019 年;Han 等人，2004 年）。该双链体的一条链（代表成熟的 miRNA）被掺入 miRNA 诱导的沉默复合物（miRISC）中。在 miRISC 中，miRNA 与其靶标 mRNA 配对。该过程涉及 6 至 8 nt miRNA 种子序列，该序列与靶 mRNA 的 3' UTR 内的互补核苷酸结合，导致翻译抑制或去烯基化和降解（O'Brien 等人，2020 年;Catalanotto等人，2016）。与 miRNA 直接相互作用的 Argonaute （AGO）蛋白和 182 kDa 的甘氨酸色氨酸蛋白（GW182）是 miRIS 组装和功能的重要因子，代表了 miRNA 抑制途径的关键步骤。在miRNA成熟过程中，Drosha和Dicer都受到几种辅因子或辅助蛋白的帮助，例如SMAD核相互作用蛋白（SNIP1）和腺苷脱氨酶（ADAR）（Eulalio等人，2009;Krol等人，2010）。miRISC 的主要成分包括 miRNA、miRNA 的 mRNA 靶标、GW182 和 AGO2。AGO2 蛋白与 miRNA 的 5' 末端的 U 或 A 结合，并在介导 mRNA：miRNA 对的形成中起重要作用，随后是 mRNA 靶标的翻译抑制或降解（O'Brien 等人，2020 年）。

miRNA loading

Both the process of MVB/exosome biogenesis and miRNA-sequence-specific determinants may modulate miRNA sorting into exosomes. As discussed earlier, ESCRT plays an important role in MVB biogenesis and exocytosis, but knockdown of ESCRT proteins does not affect miRNA sorting to exosomes (Kosaka et al., 2010). Interestingly, knockdown of Alix, an ESCRT-III accessory protein, did not affect the number of released EVs but induced a decrease in secreted miRNAs (Iavello et al., 2016). Within exosomes, the miRNAs contain common sequences, or EXO-motifs, that facilitate binding to RNA-binding proteins (RBPs), such as heterogeneous nuclear ribonucleoproteins (hnRNPA2B1) and SYNCRIP (Villarroya-Beltri et al., 2013; Santangelo et al., 2016). Some studies demonstrate that short sequence motifs overrepresented in miRNAs, such as GGAG and UGCA found in miR-198 and miR-601, control sorting into exosomes, and directed mutagenesis of these motifs modulates miRNA cargoes (Villarroya-Beltri et al., 2013).

Recent studies have also shown the connections between AGO2 and exosomal miRNA sorting, and AGO2 has been identified in exosomal protein analyses by mass spectrometry (MS) or western blotting (Zhang et al., 2015a; Goldie et al., 2014; Melo et al., 2014). It is known that knockout of AGO2 decreases the types or abundance of preferentially exported miRNAs in HEK293T-derived exosomes (Guduric-Fuchs et al., 2012). Other evidence also shows the relationship between miRISC and exosomal miRNA sorting. Thus, YBX1 (Y-box protein I), but not AGO2, binds to miR-223 and miR-144 and can control packaging into vesicles (Ung et al., 2014; Shurtleff et al., 2016). YBX1 also co-localizes with members of the RISC, including GW182, which can be found in exosomes (Goodier et al., 2007; Gallois-Montbrun et al., 2007). Thus, the main components of miRISC are co-localized with MVBs (Gibbings et al., 2009; Wang et al., 2019). In summary, specific sequences present in miRNAs may guide their incorporation into exosomes, whereas other proteins may control the sorting of exosomal miRNAs in a sequence-independent fashion.

Exosomal miRNA secretion

The proportion of miRNAs is higher in exosomes than in their parent cells, and several studies show that miRNAs are not randomly incorporated into exosomes (Guduric-Fuchs et al., 2012; Li et al., 2012; Zhao et al., 2019). Thus, a key functional point is that analysis of miRNA expression levels in various cell lines and their secreted exosomes shows that the miRNA content of cells compared to their exosomes is quite different. For example, Guduric-Fuchs et al. have shown that miR-150, miR-142-3p, and miR-451, among others, preferentially enter exosomes. In this context, it should be noted that the cellular content of miRNAs represents a mixture of endogenous miRNAs, as well as the miRNAs that enter the cell via exosomes derived from other cell types (Guduric-Fuchs et al., 2012). In this way, the miRNA cargo of a secreted exosome that enters the circulation from one cell type can significantly affect the miRNA profile of a recipient cell. It would be of interest to determine whether miRISC loading and mRNA translational repression are the same or different for endogenously produced miRNAs versus those that enter the cells through uptake of exosomes.

Mechanisms of miRNA action and target identification

As mentioned earlier, miRNAs inhibit the translation of target mRNAs within the RISC, where they bind to RPBs such as Argonaute (Ciafrè and Galardi, 2013). Through seed sequence binding to the mRNA 3′ UTR region, target mRNAs are loaded into the RISC, where translational arrest can occur (Gu et al., 2009). While the seed sequence interactions with mRNA 3′ UTR regions apply generally to miRNA/target mRNA pairs, exceptions to these rules have been demonstrated (Didiano and Hobert, 2008; Bartel, 2009; Moore et al., 2015). In some cases, miRNA sequences outside of the seed sequence or mRNA nucleotides not within the mRNA 3′ UTR are involved in the binding interaction.

miRNA sequencing studies demonstrate that exosomes harvested from blood or conditioned media (CM) of specific cell types contain hundreds of different miRNAs (Bhome et al., 2018; Van den Brande et al., 2018). However, the number of miRNA transcripts per exosome particle can be quite small given the very large number of exosomal particles in blood or CM (Bhome et al., 2018). If a particular exosomal preparation exerts a metabolic effect, it can be a daunting task to identify the individual miRNA or group of miRNAs responsible for the measured biologic effect. However, strategies for identifying the miRNA(s) of interest have been used. First, one needs to thoroughly sequence all the miRNAs within the exosomal preparation. While hundreds may be detected, most of these miRNAs are expressed at very low levels and are unlikely to exert biologic effects when taken up by recipient cells. Thus, the more abundant miRNAs are most likely to contain the miRNA(s) of interest (Yáñez-Mó et al., 2015). Since miRNAs generally downregulate their mRNA targets, it is reasonable to assume that the miRNA(s) of interest that lead to a given biologic effect will exhibit differential increased expression in exosomes derived from the cell type of interest (e.g., cells from obese animals compared to lean controls) (Veziroglu and Mias, 2020; Sigismund et al., 2012). This strategy allows one to focus on the more highly expressed exosomal miRNAs that are also differentially expressed in the condition of interest. This greatly reduces the total number of candidate miRNAs, and if the biologic effect of interest can be reduced to a cell-based screening assay, then all the candidate miRNAs can be screened for activity. This provides a methodology to identify the key miRNA, or group of miRNAs, responsible for the biologic effect observed.

If one identifies an exosomal miRNA that produces the desired biologic effect, then the next step is to define the mechanism. miRNAs exert their biologic effects by inhibiting the translation of their target mRNAs, but identifying the target mRNA for a specific miRNA can be difficult. However, strategies have been proposed that can often lead to tangible results. Using various bioinformatic tools such as TargetScan and PicTar, one can identify all the mRNA targets that could theoretically interact with a given miRNA seed sequence. This typically yields a relatively large number (dozens or more) of theoretical mRNA targets for a given miRNA. One can then use transcriptome assessments such as RNA sequencing (RNA-seq) to identify the mRNAs suppressed by a given miRNA in the target tissue. Since not all theoretical mRNA targets will be expressed in the target tissue of interest, and only a subset will be downregulated by applying the miRNA, this approach can reduce the number of potential mRNA targets to be considered. With luck, only a relatively small number of potential mRNA targets will fulfill these criteria. These can then be studied on an individual basis to identify the biologically relevant mRNA target of a given miRNA.

Due to the proximity of RBPs, miRNAs, and target mRNAs within the RISC, another general approach involves immunoprecipitation methods, which can be used to successfully identify miRNA/mRNA target pairs (Thomson et al., 2011). As a general approach, whole-cell lysates can be crosslinked with various reagents to stabilize RBP/miRNA/mRNA interactions. The RBPs (e.g., Argonaute 1) can then be immunoprecipitated, followed by reversal of the crosslinks and sequencing of the precipitated mRNAs and miRNAs (Lin and Miles, 2019; Wessels et al., 2019). With this approach, one can match the miRNAs to target mRNAs by assessing miRNA (usually seed sequence) complementarity to potential mRNA targets (Helwak et al., 2013; Nussbacher and Yeo, 2018; Van Nostrand et al., 2016).

Non-miRNA exosome cargo

Although less well studied than exosomal miRNAs, different types of proteins such as enzymes, hormones, cytokines, ligands, and others can be components of exosome cargo released from metabolic tissues and can be functional in recipient cells. For example, hypoxic adipocytes release exosomes containing enzymes related to de novo lipogenesis (Sano et al., 2014). Also, hepatocyte-derived exosomes can contain active arginase-1 that modulates the metabolism of circulating arginine, regulating vascular function (Royo et al., 2017).

The secretion of adiponectin from adipocytes can also include exosome-mediated pathways. Eicosapentanoic acid (EPA) and docosahexaenoic acid (DHA) treatment increase adiponectin in the exosome fraction (DeClercq et al., 2015), and exosomes isolated from mouse serum were associated with adiponectin. However, the proportion of total circulating adiponectin in exosomes is relatively small, suggesting that adiponectin within exosomes may not have robust biologic effects. Another hormone found in adipocyte exosomes is resistin. Rong et al. showed that melatonin decreases exosomal trafficking of resistin from adipocytes to hepatocytes, mitigating ER stress-induced hepatic steatosis (Rong et al., 2019).

Exosomal lipid cargo

Exosomal lipids can also be functional in the progression of metabolic disease. For example, sphingosine 1-phosphate (S1P) contained in endothelial-derived exosomes can enhance migration of hepatic stellate cells (HSCs) (Wang et al., 2015a). Interestingly, circulating exosomes containing C16:0 ceramide- and S1P-enriched lipid species are progressively increased in the plasma of obese patients with simple steatosis and non-alcoholic steatohepatitis (NASH) patients with early fibrosis (Kakazu et al., 2016). In addition, adipocytes can release exosome-sized, lipid-filled vesicles that become a source of lipids in local macrophages (Flaherty et al., 2019).

Adipose tissue exosome biology

Increased adipose tissue mass is a hallmark of obesity and is present in the great majority of individuals with type 2 diabetes mellitus (T2DM) (Kusminski et al., 2016; Ghaben and Scherer, 2019). In obesity, both in the presence and absence of diabetes, chronic tissue inflammation, particularly in adipose tissue and liver, is an important contributor to insulin resistance (Hotamisligil and Erbay, 2008; Lackey and Olefsky, 2016; Lee and Olefsky, 2021; Saltiel, 2021; Ferrante, 2013). Obesity-induced chronic inflammation is not confined to adipose and liver since there are reports of proinflammatory pathway activation in the CNS, the gastrointestinal tract, muscle, and pancreatic islets, consistent with an intra- and interorgan network of crosstalk in these complex heterogeneous disorders (Lee and Olefsky, 2021). A sizable literature exists exploring various cytokines, adipokines, lipid species, and other factors released from adipose tissue that may contribute to systemic metabolic dysfunction (Lago et al., 2007; Hernandez et al., 2021; Vegiopoulos et al., 2017).

Since exosomes can be released from adipose tissue and work locally in a paracrine manner or enter the circulation to have systemic effects, they have been increasingly studied as effectors of intercellular signaling. As discussed earlier, exosomes contain a variety of cargo components, and many of the biologic effects of these particles have been attributed to their miRNA content (Figure 3). While evaluation of adipose tissue vesicles in the blood is still limited by the absence of adipocyte-derived specific markers, highly expressed adipocyte proteins such as aP2/FABP4, perilipin-1, adiponectin, or PPAR have been identified in circulating EVs (Connolly et al., 2018). Nonetheless, their use as specific adipocyte markers can be confounded because their expression varies according to adipocyte differentiation state and/or hypertrophy, and many of these proteins are also expressed in adipose tissue macrophages (ATMs). Using a fat-specific knockout of the miRNA-processing enzyme Dicer, Thomou et al. showed that adipocytes were major contributors to circulating exosomal miRNAs (Thomou et al., 2017) (Figure 4A). Conversely, tracing plasma EVs in mice expressing a fluorescent protein (TdTomato) specifically in adipocytes, Flaherty et al. normalized the abundance of the fluorescent protein level of plasma exosomes to the tetraspanin CD63 and suggested that adipocyte-derived exosomes represented a minority of the circulating exosomes (Flaherty et al., 2019). Further work will be needed to evaluate the exact contribution of adipocyte vesicles to the circulating pool of EVs (Amosse et al., 2018).

In studies using whole blood as a source of circulating exosomes, the overall findings show that exosomes from obese subjects produce glucose intolerance when administered to lean mice and miRs 122, 192, 27a-3p, and 27b-3p have been implicated in these effects (Castaño et al., 2018; Párrizas et al., 2015; Jones et al., 2017) (Figure 4A). In some cases, mimics of these miRNAs reproduced the effects on glucose tolerance. Castaño et al. also showed that circulating exosomes from obese mice promoted glucose intolerance and insulin resistance when administered to lean mice (Castaño et al., 2018).

Whole adipose tissue exosomes

There are a number of studies showing that exosomes derived from both human and mouse obese adipose tissue can inhibit insulin sensitivity (Kita et al., 2019; Dang et al., 2019; Pan et al., 2019; Zhang et al., 2015b). Exosomes prepared from obese human adipose tissue exhibit differential expression of a variety of miRNAs that influence insulin signaling. For example, exosomes harvested from obese adipose tissue explants can directly promote insulin resistance (Kranendonk et al., 2014), and in one of these studies, miR-141-3p appeared to play a causative role (Dang et al., 2019). Other studies using exosomes derived from adipose tissue explants from obese mice or humans have also shown biologic effects to impair insulin sensitivity (Zhang et al., 2015b). Similar effects of exosomes from obese adipose tissue were reported by Kranendonk et al. (2014). Pan et al. found that miR-34a was upregulated in visceral fat of obese subjects and observed that knockout of miR-34a in mice protected them from obesity-induced glucose intolerance and insulin resistance (Pan et al., 2019). They further reported that miR-34a expression led to decreased ATM content in obese adipose tissue with an increase in the percentage of M2-like and a decrease in M1-like ATMs, indicating a potential mechanism for the insulin-sensitizing effects of this exosomal miRNA. Deng et al. also showed that exosome-like particles from obese mouse adipose tissue can be taken up by circulating monocytes and activate the subsequent proinflammatory differentiation program once they differentiate into macrophages (Deng et al., 2009; Flaherty et al., 2019) (Figure 4A). Using human adipose tissue explants, Ferrante et al. found that exosomes obtained from obese versus lean subjects contain a number of miRNAs that were differentially expressed (Ferrante et al., 2015). They found that a subset of these miRNAs modulated cellular signaling pathways relevant to insulin action. However, it is important to note that adipose tissue is composed of a variety of different cell types (adipocytes, macrophages, immune cell types, and ECs), and therefore, when studying adipose tissue explant-derived exosomes, the specific cell type producing the biologically relevant exosomes cannot be determined.

Adipocyte exosomes

Using adipocytes obtained from obese animals or high glucose-treated 3T3L1 adipocytes, the harvested adipocyte exosomes directly induce monocyte differentiation into inflammatory macrophages (Eguchi et al., 2015). In a similar vein, Pan et al. found that exosomes derived from adipocytes can inhibit the M2-like macrophage polarization state, and this effect was attributed to miR-34a (Pan et al., 2019) (Figure 4A). Thomou et al. used their adipocyte-specific dicer knockout mice to assess the biologic effects of adipocyte-derived exosomes (Thomou et al., 2017). They found that exosomes released from brown adipocytes express high levels of miR-99b, which can enter the circulation and travel to the liver, where they inhibit FGF21 expression. It is possible that corresponding changes in blood levels of FGF21 have metabolic consequences in obesity and/or T2DM. Adipose-derived exosomal miRNAs also have paracrine functions as exosomes released from large adipocytes contain miR-16, miR-27a, miR-146b, and miR-222, which can be transferred to small adipocytes to stimulate lipogenesis and adipocyte hypertrophy (Müller et al., 2011) (Figure 4A).

Adipocyte exosomes can also contain non-miRNA cargoes that exert distant biologic effects. For example, Crewe et al. show that exosomes produced by adipocytes from obese mice contain mitochondrial particles and mitochondria proteins that exhibit oxidative damage but still display respiratory function. These exosomes can be released from adipocytes and taken up by cardiomyocytes in the heart where they trigger production of reactive oxygen species (ROS). This exerts protective effects in the heart against oxidative stress and ischemia/reperfusion injury. In this manner, adipocyte-derived exosomes in obesity can actually exert cardioprotective effects in certain circumstances (Crewe et al., 2021). Exosomes themselves are heterogeneous in terms of size, cargo content, and cell origin, and with current techniques, it is difficult to make a “pure” population of exosomes. While highly enriched exosome preparations can be produced, it is possible that some of the reports in the exosome/metabolism field include studies of preparations of exosomes heavily mixed with other EV subtypes.

Adipose tissue macrophage exosomes

Obesity is characterized by a substantial accumulation of ATMs. These ATMs have been denoted as either classically activated proinflammatory M1 or alternatively activated anti-inflammatory M2 cells. However, the terms M1 or M2 do not adequately apply to in vivo macrophage populations such as ATMs. These labels relate to bone marrow-derived macrophages (BMDMs) that have been polarized in vitro by treatment with either LPS to generate M1 BMDMs or IL-13/IL-4 to create M2 BMDMs. Macrophage populations in vivo may have similarities to the M1 or M2 state, but they are not the same (Li et al., 2019a; Xiong et al., 2019; Jaitin et al., 2019; Seidman et al., 2020; Zhao et al., 2017). Detailed bulk RNA-seq or single-cell RNA-seq studies have demonstrated that there are discrete clusters of macrophage subpopulations within these general categories (Li et al., 2019a; Xiong et al., 2019; Jaitin et al., 2019; Seidman et al., 2020; Zhao et al., 2017). Recent papers often use the phrases M1-like or M2-like to indicate this concept when describing in vivo macrophage populations. In obesity, the increased number of ATMs involves an accumulation of proinflammatory M1-like cells (Boutens and Stienstra, 2016). These macrophages produce proinflammatory cytokines, ROS, and nitric oxide (NO), which all play a role in eliminating exogenous pathogens (Lee and Olefsky, 2021).

Many studies have shown that these M1-like ATMs are major contributors to the insulin-resistant state (Russo and Lumeng, 2018; Hill et al., 2014; Hotamisligil and Erbay, 2008; Lackey and Olefsky, 2016; Lee and Olefsky, 2021; Saltiel, 2021; Ferrante, 2013) (Figure 4A). This has prompted studies of ATM-derived exosomes. Thus, Fuchs et al. showed that plasma- and adipose tissue-derived exosomes from obese subjects caused decreased insulin signaling in myotubes and hepatocytes (Fuchs et al., 2021). Furthermore, exosomes harvested from ATMs derived from obese mice directly cause insulin resistance in vitro when incubated with adipocytes, primary hepatocytes, or L6 myocytes (Ying et al., 2017). These “obese” ATM exosomes were also given intravenously to chow-fed lean mice. Interestingly, these “obese” exosome-treated lean recipient mice developed glucose intolerance, hyperinsulinemia, and insulin resistance comparable to that observed in obese mice (Ying et al., 2017). Since body weight and food intake were unchanged by in vivo exosome treatment, these findings indicate that “obese” ATM exosomes can directly cause insulin resistance. Another study by Liu et al. reported that miR-29a was a key pathogenic RNA within obese ATM exosomes causing insulin resistance (Liu et al., 2019). In contrast, ATM exosomes derived from lean mice led to the opposite phenotype (Ying et al., 2017). Thus, treatment of insulin target cells (adipocytes, myocytes, or primary hepatocytes) with “lean” ATM exosomes directly caused enhanced insulin signaling with an improvement in overall cellular insulin sensitivity. In vivo treatment of insulin-resistant, hyperglycemic obese mice with “lean” ATM exosomes resulted in improved glucose tolerance with enhanced insulin sensitivity. Overall, one could view these “lean” ATM exosomes as an effective therapeutic modality, improving metabolic dysfunction in obesity. These studies also showed that either the “obese” or “lean” ATM exosomes exert their metabolic effects entirely through their miRNA cargo since depletion of miRNAs from these exosomes by knockout of proteins critical for processing or loading of miRNAs into exosomes, such as Drosha or YBX1, abrogated all the exosome-mediated effects. In other words, miRNA-free “lean” or “obese” exosomes did not exert metabolic effects either in vitro or in vivo.

ATMs in lean mice generally have an anti-inflammatory phenotype and are often termed alternatively activated, or M2-like. More in-depth studies of “lean” ATM exosomes are challenging due to the paucity of ATMs in the lean state. However, bone marrow-derived progenitor cells can be cultured in vitro with IL-13 and IL-4, which drives them toward a more classical M2 polarization state. Although ATMs from lean mice and M2 BMDMs are comparable, they are not identical, and certain phenotypic and transcriptomic differences exist (Li et al., 2019a). Nevertheless, a recent report shows that exosomes harvested from M2 BMDMs cause improved insulin signaling in adipocytes, myocytes, and primary hepatocytes in vitro, and when administered intravenously to obese mice, they improve glucose tolerance and enhance insulin sensitivity (Ying et al., 2021). miRNA sequencing of exosome preparations yields a relatively complete picture of all the miRNAs. Using exosomal miRNA sequencing, a bioinformatics approach, and cell-based screening assays, Ying et al. showed that miR-690 was the major miRNA component in these M2 BMDM exosomes leading to the beneficial metabolic effects (Figure 4B). Indeed, treating insulin target cells in vitro or obese mice in vivo with a synthetic mimic of miR-690 recapitulated all the favorable metabolic effects of the M2 BMDM or “lean” ATM exosomes. A synthetic antagomir of miR-690 blocked these effects, indicating the specificity of the findings. Interestingly, both “lean” ATM exosomes and M2 BMDM exosomes expressed comparably high levels of miR-690. These M2 BMDM exosomes or the miR-690 mimic also caused a robust anti-inflammatory effect in obese mice leading to a decrease in M1-like and an increase in M2-like ATMs. Similar findings were noted in vitro in which miR-690 promoted an anti-inflammatory polarization phenotype in BMDMs. These studies provide an example of exosomal miRNA biology leading to the identification of a specific miRNA, which might have future therapeutic potential as an insulin sensitizer if translated into humans.

Conceptually similar results were obtained by Zhang et al., who found that exosomes generated from adipocytes isolated from high-fat diet mice promoted macrophage differentiation to the pro-inflammatory M1-like state (Zhang et al., 2015b). These investigators attributed these effects to exosomal miR-155 (Figure 4A). Not surprisingly, once these macrophages were polarized to the proinflammatory state, CM from these cells directly caused decreased insulin sensitivity in skeletal muscle cell lines. Subsequent studies showed that macrophages activated via LPS treatment produce exosomes that inhibit insulin-stimulated glucose transport in adipocytes (De Silva et al., 2018).

Other studies in human and mouse macrophages have supported a role for M1-like macrophage-derived exosomes in promoting macrophage proinflammatory pathways. For example, in both human and mouse macrophage exosomes, miR-155 has been identified as a potent pro-inflammatory factor by repressing the expression of PPARγ, Socs1, or Socs6 (O’Connell et al., 2007; Ying et al., 2017; Wang et al., 2017; Ge et al., 2021). Chen et al. showed that LPS stimulation of the human macrophage THP-1 cell line leads to the release of exosomes that can activate the stellate cell fibrogenic program, and they implicated miR-103-3p in this process (Chen et al., 2020b). If this phenomenon also applies to hepatic macrophages during the development of hepatic inflammation and fibrosis, then this could represent a NASH-enhancing mechanism.

Endothelial cell exosomes

EC-derived exosomes are released upon activation or apoptosis. EC exosomes constitute a large subclass of all circulating vesicles in peripheral blood (5%–15%), although a sizeable proportion of circulating plasma exosomes are also derived from platelets and erythrocytes (Combes et al., 1999; Dignat-George and Boulanger, 2011; Markiewicz et al., 2013; Arraud et al., 2014). EC exosomes can impair vascular function through pro-coagulative (Combes et al., 1999) and pro-inflammatory functions (Buesing et al., 2011), as well as by mitigating NO production from ECs (Brodsky et al., 2004; Densmore et al., 2006) (Figure 4B).

Proteomic analyses showed that secreted EC exosomes contain proteins that are also found in the originating ECs, and some can be transferred into recipient cells (Liu et al., 2013). Other inflammatory factors, including interleukin-1 (IL-1), interferon-γ (IFN-γ), and bacterial LPS, can induce the release of EC exosomes, which contain specific miRNAs that were present in significantly lower amounts compared to vesicles derived from unstimulated ECs (Yamamoto et al., 2015). Crewe et al. used a cav1 mouse model to show that neighboring ECs transfer cav1-containing exosomes to adipocytes in vivo, which reciprocate by releasing exosomes taken up by ECs (Crewe et al., 2018) (Figure 4B). Because EC exosomes can contain signaling molecules derived from the circulation, one can speculate that the effect of EC exosomes on adipocyte physiology is dependent on the type of signals in the blood at any given time. This mechanism would enable transfer of plasma constituents from ECs to adipocytes and may be regulated by feeding status and obesity, suggesting that exosomes participate in the tissue response to changes in the systemic nutrient state. If so, it follows that EC exosomes could customize the functional response of adipocytes to systemic changes in metabolism. The potentially wide-ranging implications of the EC-adipocyte communication axis for adipocyte function and whole-body metabolism are still unclear (Crewe et al., 2018).

Skeletal muscle exosomes

Several studies have shown both paracrine and endocrine effects of skeletal muscle-derived exosomes in the maintenance of muscle homeostasis and communication with other tissues (Qin and Dallas, 2019; Li et al., 2019b). Muscle cells, such as myoblasts and myotubes, are a source of exosomes expressing Tsg101 and Alix protein markers, along with other proteins involved in signal transduction (Rome et al., 2019; Forterre et al., 2014; Guescini et al., 2010). As myoblasts differentiate into myotubes, the pattern of secreted proteins changes. Proteomic analysis showed that most of the exosomal proteins are derived from skeletal muscle cytosol. Of these proteins, only 10% have a canonical secretory sequence, and 43% were identified as non-classical secreted proteins lacking an N-terminal secretory sequence (Safdar et al., 2016). Exosomes may play a role in skeletal muscle differentiation and maturation as many proteins involved in myoblast to myotube formation are found in exosomes (Le Bihan et al., 2012).

Emerging evidence suggests that exercise enhances the biogenesis of MVBs and the resulting exosomes (Garner et al., 2020). Subsequently, exosome release following exercise can carry exerkines that might modulate intercellular communication (Safdar and Tarnopolsky, 2018). Several papers have suggested that these might modulate aging (Bertoldi et al., 2018), T2DM (Li et al., 2019b; Safdar et al., 2016), cardiovascular diseases (CVDs) (Bei et al., 2017; Zhou et al., 2019; Ma et al., 2018), immunity (Lancaster and Febbraio, 2005; Wu and Liu, 2018), and sarcopenia (Rong et al., 2020).

It is known that palmitate-induced skeletal muscle insulin resistance triggered the release of a new population of myotube-derived exosomes enriched in palmitate. (Aswad et al., 2014). Palmitate found in myotube-derived exosomes from diet-induced obese (DIO) mice was transferred to recipient myotubes and resulted in the upregulation of genes involved in cell cycle and cellular adhesion (Figure 4C). Among them, the pro-inflammatory cytokine IL-6 and the cell-cycle regulator cyclin D1 were strongly upregulated, whereas Glut4 was downregulated.

Exosomes and beta cell function

Exosomes have been implicated in regulating pancreatic beta cell function. For instance, skeletal muscle-derived exosomes from mice fed with a standard diet enriched with 20% palmitate induced the proliferation of the beta cell MIN6B1 cell line as well as in isolated mouse islets (Marchand et al., 2016). These skeletal muscle-derived exosomes were enriched in miR-16 that was transferred to the MIN6B1 cells and regulated Ptch1, a gene involved in pancreas development (Marchand et al., 2016) (Figure 4C).

Several studies have also shown potential beneficial effects of MSC-derived exosomes on beta cells. Intravenous injections of MSC-exosomes reduced fasting blood glucose levels, restored basal insulin levels (Sabry et al., 2020), and promoted beta cell regeneration, improving insulin secretion (Mahdipour et al., 2019) in diabetic rats. Furthermore, MSC-exosomes protected beta cells against hypoxia-induced apoptosis via enriched exosomal miR-21 (Chen et al., 2020a).

Beta cells also secrete exosomes in response to stimuli or stress and can affect neighboring cells. Guay et al. have shown that exosomes secreted from MIN6B1 and INS-1 can efficiently transfer miRNA to MIN6B1 recipient cells (Guay et al., 2015). Furthermore, exosomes from cytokine-treated MIN6B1 cells induce apoptosis in the recipient MIN6B1 cells mediated by AGO2 (Guay et al., 2015). Lakhter et al. showed that cytokine-treated beta-cell lines and human islets led to enhanced secretion of exosomal miR-21-5p (Lakhter et al., 2018) (Figure 4D). While beta-cells clearly secrete exosomes that can work locally in a paracrine manner on neighboring beta-cells, whether beta-cell exosomes can enter the circulation to produce systemic effects is problematic since their contribution to the total circulating pool of exosomes would be negligible.

Exosomes in NAFLD and NASH

Non-alcoholic fatty liver disease (NAFLD) is an extremely common feature of obese and T2DM individuals, and ∼30% of these subjects ultimately develop NASH. NASH is associated with increased risk for CVD and is a precursor condition to cirrhosis and hepatocellular cancer. Within the liver, intercellular communication plays an important role in the progression of inflammation and fibrosis, which are major hallmarks of NASH. In the NASH liver, hepatocytes exhibit steatosis and lipotoxicity, Kupffer cells (KCs) and recruited hepatic macrophages (RHMs) promote liver inflammation, and HSCs are the primary drivers of fibrosis. All liver cells, including hepatocytes, HSCs, hepatic macrophages, and liver sinusoidal ECs, are capable of exosome release (Sung et al., 2018), suggesting that paracrine-mediated intercellular crosstalk between liver cell types could be an important contributing mechanism to hepatic disease.

In healthy conditions, hepatocytes produce exosomes containing sphingosine kinase 2, contributing to cell survival, growth, and proliferation (Nojima et al., 2016). In addition, hepatocyte-derived exosomes can mediate other biologic effects through their miRNA cargo. For example, in the early stages of obesity, hepatocyte exosomes are enriched in miR-3075, which can produce robust insulin-sensitizing effects both in vitro and in vivo when given to obese mice. A major target for miR-3075 is FA2H, which participates in the production of ceramides. Since FA2H is suppressed by miR-3075, decreased ceramide levels may provide at least part of the mechanism for the insulin-sensitizing effects of this miRNA. Interestingly, in chronic obesity, hepatocyte exosomes no longer carry appreciable amounts of miR-3075 and do not have insulin-sensitizing effects. Indeed, hepatocyte-derived exosomes from chronic obese mice promote insulin resistance by stimulating proinflammatory activation and polarization of macrophages. In this way, the initial response of hepatocytes is to produce insulin-sensitizing exosomes, and this might be viewed as a compensatory phenomenon to mitigate the full development of insulin resistance in obesity. As time goes on and chronic obesity ensues, this compensatory response is lost and hepatocyte exosomes promote insulin resistance through their proinflammatory actions (Yudong Ji et al., 2021).

Hepatocytes can also release exosomes that modulate the transcriptional program of neighboring hepatocytes and non-parenchymal cells toward inflammation and fibrosis (Hernández et al., 2020). One of the key events in NAFLD progression is the accumulation of potential harmful lipids in hepatocytes, leading to hepatocyte lipotoxicity (Alkhouri et al., 2009). Liver lipotoxicity increases hepatocyte exosome release, which might contribute to inflammation and fibrosis (Figure 4E). For example, lipotoxicity-induced hepatocyte exosomes can stimulate the expression and secretion of pro-inflammatory cytokines, contributing to local inflammation (Hirsova et al., 2016). Another study showed that lipid-induced hepatocyte exosomes are released in a caspase-3-dependent manner and promote EC activation (Povero et al., 2013). These exosomes also stimulated HSC activation via delivery of miR-128-3p that suppresses PPAR-γ expression (Povero et al., 2015). In addition, exosomes carrying miR-1 were released from steatotic hepatocytes and mediated pro-inflammatory effects in ECs via downregulation of KLF4 and activation of the NF-κB pathway (Jiang et al., 2020). Lipotoxicity-induced hepatocyte exosomes are enriched in chemokine (C-X-C motif) ligand 10 (CXCL10), which is a macrophage chemoattractant (Ibrahim et al., 2016). This results in recruitment and M1-like polarization of hepatic macrophages (Tomita et al., 2016), as well as monocyte adhesion (Dai et al., 2020). Moreover, hepatocyte exosomes can contribute to hepatic recruitment of monocyte-derived macrophages by promoting monocyte adhesion via integrin β1-dependent mechanisms (Guo et al., 2019). Thus, hepatocyte exosomes can activate KCs and promote the recruitment and activation of RHMs during NASH development (Figure 4E).

Data from different diet-induced animal models of NASH have shown that blood exosome concentration increases with disease progression in a time-dependent manner (Kakazu et al., 2016; Povero et al., 2013). These exosomes can amplify inflammation through multiple mechanisms such as macrophage activation and monocyte chemotaxis (Hernández et al., 2020). The release of hepatocyte-derived exosomes was reduced by inactivating the DR5 signaling pathway or inhibiting Rho-associated protein kinase 1 (ROCK1), suggesting that ROCK1 inhibition in NASH mice could lead to a reduction in exosome release with less liver inflammation and fibrosis (Hirsova et al., 2016).

More recently, human liver stem cell (HLSC)-derived exosomes have been used to treat mice with diet-induced steatohepatitis (Bruno et al., 2020). Treatment significantly downregulated hepatic profibrotic and pro-inflammatory gene expression and ameliorated the histological abnormalities in mice with NASH. Proteomic analysis of sinusoidal-derived exosomes identified various anti-inflammatory proteins, which may have contributed to the observed beneficial effects (Bruno et al., 2020).

Several reports have shown that lipotoxic hepatocytes can release exosomes that stimulate activation and proliferation of HSCs, with increased expression of a variety of genes in the fibrotic pathway. Exosomal miRNAs 28-3P and 192 have been implicated in these effects. Interestingly, exosomes derived from lipotoxic hepatocytes can express Vanin-1 on the surface, and treatment with Vanin-1 neutralizing Abs prevents exosome-mediated HSC activation. HSCs themselves are another important source of exosomes that contribute to liver injury in NASH. Activation or trans-differentiation of HSCs results in insoluble collagen deposition and distortion of the normal macro- and micro-anatomical structures of the liver (Friedman, 2008). During liver injury, exosomes from activated HSCs containing the pro-fibrogenic connective tissue growth factor (CTGF) can activate other HSCs (Charrier et al., 2014). Additionally, intercellular communication between quiescent and activated HSCs via exosomes may also modulate fibrosis since activated HSCs can release exosomes that activate quiescent HSCs. In this regard, a role for miR-214 in regulating the expression of alpha-smooth muscle actin and collagen in activated HSCs has been demonstrated (Chen et al., 2015).

In summary, intercellular signaling among hepatocytes, KCs, RHMs, and HSCs plays a key role in the progression from NAFLD to NASH, and local communication through exosomes may be integral to this process. Despite the abundant evidence of intercellular communication within the liver, it should be pointed out that there is also evidence that signals extrinsic to the liver have an important impact on NAFLD and NASH. This could include cytokines and adipokines from obese adipose tissue, signals from a leaky gastrointestinal tract such as LPS, and exosomes originating outside the liver, all of which can travel to the liver through the circulation, promoting NAFLD and NASH.

Exosomes as biomarkers for metabolic disease

With respect to biomarker studies, exosomal cargo represents a way to sample the metabolic state within cells at a given point in time. With this line of reasoning, circulating exosomal cargo can be thought of as a liquid biopsy of the metabolic state of the originating cell type. In the best case, a biomarker would have specificity for the etiology or complications of a particular disease. With respect to obesity, while it is not necessary to utilize a circulating biomarker to diagnose the obese state, it would be of high importance to identify biomarkers that might predict the development of obesity, weight loss success, or recidivism. To the extent that biomarker studies focus on circulating exosomes, an important caveat must be kept in mind. The circulating exosomal pool is the composite of all exosomes released from a large variety of different cell types. Therefore, depending on the disease in question, exosomal signatures from the relevant disease-related cell types may be blunted or confounded by the presence of circulating exosomal cargo derived from multiple other cells or tissues. In addition, when focusing on exosomes as biomarkers, another important consideration is that many of the earlier studies did not use current methods to produce well-characterized enriched exosome preparations, so some of the reported miRNA signatures may reside to an unknown extent in non-exosomal EV components.

Previous studies have shown that changes in circulating miRNA profiles are one of the physiological responses to the development of metabolic diseases (Hsieh et al., 2015; Nunez Lopez et al., 2016; Ortega et al., 2013; Pescador et al., 2013; Villard et al., 2015; Wang et al., 2015b; Willeit et al., 2017; Wu et al., 2015). Thus, the potential of circulating miRNAs as signatures for metabolic diseases has been studied in both humans and animal models. In evaluating biomarker studies of this type, it is important to distinguish whether the miRNAs of interest are within exosomes, bound to blood proteins, or circulating free in the circulation. Among a group of differentially expressed miRNAs, Ortega et al. found that miR-140-5p abundance was greater in the blood of morbidly obese patients (BMI ≥ 40 Kg/m²) than obese men (40 Kg/m² > BMI ≥ 30 Kg/m²) (Ortega et al., 2013). After surgery-induced weight loss, circulating miR-140-5p was markedly reduced, suggesting that this miRNA could be a biomarker for the change of fat mass. In addition, circulating miRNAs could be predictive signatures suggesting which obese patients are at greatest risk for T2DM development. In the population-based Malmӧ Diet and Cancer Study Cardiovascular Cohort, Gallo et al. reported that circulating miR-483-5p level was associated with the incidence of T2DM and CVD (Gallo et al., 2018). In addition, numerous studies have reported changes in circulating miRNA profiles associated with obesity or other metabolic diseases (Nunez Lopez et al., 2016; Wang et al., 2015b; Willeit et al., 2017; Wu et al., 2015). However, very few miRNA candidates were concordant across these studies, and different patterns were observed. These inconsistent results may relate to technical differences, and some studies have assessed all circulating miRNAs, while others have assessed EV miRNAs, with only a few examining exosomal miRNAs, and the miRNA cargo among these different components is different. In addition, the methods of miRNA measurements are not uniform, with some reports using chromatin immunoprecipitation (ChIP)-based technologies and others using direct sequencing to assess the miRNA expression signatures. Interestingly, an increase in the absolute level of circulating EVs in obesity has been a consistent observation in several studies.

Ghai et al. reported a set of miRNAs that are significantly decreased in plasma exosomes of T2DM patients after metformin treatment (Ghai et al., 2019). More importantly, among these metformin-repressed exosome miRNAs, most were expressed at greater levels in treated T2DM patients than in healthy humans. In contrast, these metformin-mediated changes in miRNA abundance were barely detected in whole plasma or exosome-depleted plasma. Another early study by Povero et al. found that in choline-deficient diet-induced liver steatosis, most of the circulating miR-122 was contained within exosomes, while in lean control mice, miR-122 was mostly bound to Argonaute 2 in the circulation independent of exosomes (Povero et al., 2014). In addition, Endzeliņš et al. suggest that miRNAs within plasma exosomes could be a more predictable source of miRNAs than whole plasma (Endzeliņš et al., 2017). Exosomes can be secreted by most cell types, and it has been a challenge to distinguish the cell-specific origin of exosomes and their miRNA cargos in biofluids. Emerging evidence indicates that some exosomes harbor cell-specific surface proteins that allow precipitation from biofluids. For example, Prattichizzo et al. found that both platelet and activated EC-derived exosomes carry CD31, allowing them to enrich CD31+ exosomes from human plasma using a CD31 antibody-conjugated magnetic bead-based precipitation method (Prattichizzo et al., 2021). They found that a group of specific miRNAs within CD31+ plasma exosomes from T2DM patients are tightly associated with the incidence of adverse cardiovascular events. Although many studies have profiled miRNAs within exosomes isolated from CM after ex vivo culture of primary cells, these exosome miRNA profiles may differ from those expressed under physiological conditions in vivo. Thus, methods to more precisely measure cell-specific circulating exosome miRNAs will greatly advance this area of the biomarker field. Taken together, these findings support the concept that circulating miRNAs could be sensitive signatures for metabolic diseases, but future studies will be required to validate this concept.

Exosome protein cargo composition is also associated with certain cellular functions, suggesting that exosome protein signatures could be potential biomarkers. Thus, Freeman et al. have reported that human plasma exosomes harbored various proteins associated with insulin signaling and that lower levels of phospho-S6RP, phospho-GSK3β, and phospho-AKT within plasma exosomes were associated with greater HOMA-B levels (Freeman et al., 2018). In addition, greater HOMA-IR was significantly associated with less phospho-S6RP within plasma exosomes. Also, plasma exosomes isolated from T2DM patients contained less leptin receptor and phospho-IR than in healthy human plasma exosomes. These findings suggest that circulating exosome protein cargo may provide markers of beta cell dysfunction and/or insulin resistance. By comparing the protein profiles within plasma exosomes derived from healthy, pre-cirrhotic, and cirrhotic NASH humans, Povero et al. found a set of hepatocyte-derived exosome proteins that have potential as biomarkers for the diagnosis of NASH (Povero et al., 2020). However, the number of reports focusing on exosome proteins as biomarkers is quite limited, and future work will be necessary to explore this issue.

Translational and clinical aspects

Exosomes as therapeutics or drug delivery vehicles

Given the nature of exosomes as nanovesicles, which can transfer cargos across different biological barriers, exosomes have been explored as delivery vectors for various molecules, including proteins and different RNA species. Interestingly, some studies have found that the uptake of exosomes can be cell specific, which is an important feature for drug delivery (Hoshino et al., 2015, 2020). However, there is a paucity of clinical papers on this subject in metabolic diseases, although studies are now gearing up. In other fields, more significant progress has been made, and a couple of these studies will be briefly summarized as examples of what might emerge in the future in the field of metabolism. For example, Hoshino et al. suggested that tumor cell-derived exosomes harbor specific integrins, which direct exosome homing to specific organs (Hoshino et al., 2015). Indeed, an early study showed that exosomes expressing αv integrin-specific peptides efficiently deliver exosomes to αv integrin+ tumors in prostate, breast, cervical, and pancreatic cancer models (Sugahara et al., 2009). In addition, Alvarez-Erviti et al. engineered mouse dendritic cells to produce neuron-specific RVG (rabies viral glycoprotein) peptide expressing exosomes that can specifically deliver small interfering RNA (siRNA) cargos into neurons, microglia, and oligodendrocytes after intravenous injection (Alvarez-Erviti et al., 2011). Another study by Yang et al. also used RVG-expressing exosomes to deliver miR-124 into the infarction site in a mouse stroke model (Yang et al., 2017). Similar engineering approaches have been used in other studies. For example, Kooijmans et al. found that fusion of glycosylphosphatidylinositol anchor peptides enhanced the binding efficiency of exosomes harboring anti-EGFR nanobodies into EGFP-positive tumor cells (Kooijmans et al., 2016). Ongoing clinical studies are being conducted to validate these findings from animal models.

An increasing number of studies have shown the effects of exosomes in metabolic diseases. Exosomes from MSCs have been tested as therapeutics in several clinical studies because of their regenerative capacity, immune tolerance, and immunoregulatory functions. The first report of MSC-derived exosomes in humans showed a significant attenuation of acute graft-versus-host disease symptoms within the first week of MSC exosome treatment, which remained stable for 4 months (Kordelas et al., 2014). In another study, Nassar et al. found that treatment of patients with chronic kidney disease (CKD) with human umbilical cord blood-derived MSC exosomes attenuated inflammatory responses and the progression of CKD (Nassar et al., 2016). In addition, a clinical trial has been initiated to evaluate the effects of human umbilical cord blood-derived MSC exosomes on beta cell mass in type 1 diabetes patients (ClinicalTrials.gov identifier NCT02138331). However, results from this trial have not yet been reported. Another clinical trial is ongoing to evaluate the effects of ginger- or aloe-derived exosomes on insulin resistance and chronic inflammation in patients with polycystic ovarian syndrome (NCT03493984). With respect to the concept of providing human exosomes from a donor to a recipient, unwanted immunologic reactions are unlikely since exosome-containing whole-blood and plasma transfusions have been used for decades.

Conclusions and future directions

Exosomes are nanoparticles secreted by all cell types that gain access to the interstitial space and ultimately the circulation. In this manner, exosomes represent a widespread signaling system for intercellular and interorgan crosstalk. Exosome secretion and cargo loading are regulated by nutritional inputs, cell stress, and a wide variety of physiologic or pathophysiologic states. Given these regulatory events and the ability of secreted exosomes from a given cell to act on recipient cells at distal sites, the exosomal system displays many features of classical endocrine signaling. The exosomal miRNA cargo can affect a variety of biologic pathways, and new studies appear on a regular basis identifying specific miRNAs, or groups of miRNAs, that regulate metabolic events. Undoubtedly new and improved methods will emerge to generate more highly purified exosome preparations, which will be useful to address outstanding questions concerning the regulation of exosome secretion, targeting of exosomes to specific destinations, and detailed mechanisms of exosome uptake by recipient cells. The use of circulating exosomes as a liquid biopsy method to identify disease biomarkers has already yielded positive results, and future studies are likely to identify improved biomarker signatures, which will prove clinically useful. Exosomes have substantial therapeutic potential, and clinical studies outside the field of metabolism are already underway. In the field of metabolism, future efforts could involve the administration of natural exosomes, artificial exosomes containing specific miRNA cargos, as well as other types of payloads loaded into exosomes. The field of exosome biology is moving at a rapid pace, and one can envision answers to some of the important questions in the near future.