Adipose Tissue as a Major Launch Spot for Circulating Extracellular Vesicle-Carried MicroRNAs Coordinating Tissue and Systemic Metabolism

Citation: Diez-Roda, P.; Perez-Navarro, E.; Garcia-Martin, R. Adipose Tissue as a Major Launch Spot for Circulating Extracellular Vesicle-Carried MicroRNAs Coordinating Tissue and Systemic Metabolism. Int. J. Mol. Sci. 2024,25, 13488. https://doi.org/10.3390/ ijms252413488 Academic Editor: Jung-Hyun Lee Received: 25 November 2024 Revised: 7 December 2024 Accepted: 11 December 2024 Published: 17 December 2024 Copyright: © 2024 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). Review Adipose Tissue as a Major Launch Spot for Circulating Extracellular Vesicle-Carried MicroRNAs Coordinating Tissue and Systemic Metabolism Paula Diez-Roda †, Elena Perez-Navarro †and Ruben Garcia-Martin * Department of Immunology and Oncology, Centro Nacional de Biotecnología (CNB)-CSIC, 28049 Madrid, Spain; [email protected] (P.D.-R.); elena.per[email protected] (E.P.-N.) *Correspondence: r[email protected] †These authors contributed equally to this work. Abstract: Circulating microRNAs (miRNAs), especially transported by extracellular vesicles (EVs), have recently emerged as major new participants in interorgan communication, playing an important role in the metabolic coordination of our tissues. Among these, adipose tissue displays an extraordinary ability to secrete a vast list of EV-carried miRNAs into the circulation, representing new hormone-like factors. Despite the limitations of current methodologies for the unequivocal identification of the origin and destination of EV-carried miRNAs in vivo , recent investigations clearly support the important regulatory role of adipose-derived circulating miRNAs in shaping the metabolism and function of other tissues including the liver, muscle, endocrine pancreas, cardiovascular system, gastrointestinal tract, and brain. Here, we review the most recent findings regarding miRNAs transported by adipose-derived EVs (AdEVs) targeting other major metabolic organs and the implications of this dialog for physiology and pathology. We also review here the current and potential future diagnostic and therapeutic applications of AdEV-carried miRNAs. Keywords: adipose tissue; microRNAs; extracellular vesicles; exosomes; metabolism; intercellular communication 1. Introduction Adipose tissue was traditionally observed as a long-term energy storage organ, but it is now recognized as a tissue playing a major role in the integration of systemic metabolism. This is mediated, at least in part, by its ability to secrete numerous protein factors, named adipokines [ 1 ]. These proteins have diverse regulatory functions both locally and in other tissues on insulin sensitivity, inflammation, energy balance, and many other processes [ 1 , 2 ]. However, recent research has greatly expanded the repertoire of non-protein molecules that participate in intercellular communication, representing novel adipokine-like messengers. These include several lipids (termed lipokines [ 3 ]) and RNAs, especially miRNAs. miRNAs are short non-coding single-stranded RNAs produced by virtually all cells in our body that play a pervasive role in posttranscriptional regulation, estimated to control the expression of up to 60% of our genes [ 4 ]. Transcribed as double-stranded pri-miRNAs, they are later processed by Drosha and Dicer to generate a miRNA duplex, from which one or the two strands constitute the mature miRNAs that interact with Argonaute (AGO) [ 4 ]. AGO constitutes an integral part of the miRNA-Induced Silencing Complex (RISC) that ultimately leads to the repression of mRNA targets mainly by two complementary molecular mechanisms: mRNA destabilization/decay and translational repression. Briefly, once AGO is loaded with a single-stranded miRNA, it scans the transcriptome, searching for complementary mRNA targets. In most cases, miRNA only pairs through its first 2–8 nucleotides from the 5 ′ end (called the seed region) with the mRNA target, often within the 3 ′ -untranslated region (3 ′ UTR) [ 4 , 5 ]. This leads to the recruitment of the adaptor protein TNRC6, the Int. J. Mol. Sci. 2024,25, 13488. https://doi.org/10.3390/ijms252413488 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2024,25, 13488 2 of 23 polyA-binding protein PABPC, and several deadenylases (e.g., CNOT, PAN2-PAN3, etc.), which shorten the polyA-tail of the target mRNA [ 6 , 7 ]. In addition, recruitment of DDX6 and 4E-T permits the cap-binding protein 4EHP to displace eIF4E from the 5 ′ cap, leading to mRNA decapping. Both processes (3 ′ polyA deadenylation and 5 ′ decapping) result in mRNA destabilization and decay through the action of exonucleases [ 6 , 7 ]. In addition, the dissociation of eIF4E from the cap structure impedes ribosomal assembly and scanning of the target mRNA, thus leading to translational repression [ 6 , 7 ]. Moreover, in rare cases in mammals where miRNA and target mRNA extensively pair beyond the seed region, AGO (mainly AGO2) directly slices the mRNA target, resulting in stronger repression [ 4 ]. The latter is the predominant mode of action of small interfering RNAs (siRNAs). siRNAs are short RNAs similar in size to miRNAs that, by directly incorporating into the RISC complex, also lead to the inhibition of target genes. However, siRNAs often target a unique gene by full complementarity whereas miRNAs typically exert global inhibitory effects on a given biological process by targeting multiple genes within the same or related pathway. Due to the abundance of RNases in blood and other extracellular biofluids [ 8 ], the possibility of RNAs (including miRNAs) participating in long-distance intercellular communication was never deemed as possible until the discovery of extracellular miRNA carriers. The first identifications of miRNAs as novel participants in intercellular communication were made in the late 2000s, when they were found to be carried by extracellular vesicles (EVs) and delivered to recipient cells, leading to changes in cellular function [ 9 , 10 ]. Since then, other extracellular miRNA carriers besides EVs have been identified such as circulating RNA-binding proteins (RBPs), lipoproteins, and nanoparticles [ 11 – 14 ]. While there has been extensive research on EV-carried miRNAs in interorgan communication, the contribution of these other miRNA vehicles has been barely studied. For this reason, the findings highlighted here refer to miRNAs transported in EVs. 2. Classification of EVs Every eukaryotic cell is able to release EVs to the extracellular environment. They constitute heterogenous populations of membrane particles loaded with specific repertoires of macromolecules including proteins, different RNA subtypes (miRNAs, tRNAs, snoRNAs, snRNAs, and many others), lipids, metabolites, and even organelles such as mitochondria [ 15 – 17 ]. From the different subclasses of RNA, miRNAs are among the most abundant cargoes, although the exact contribution is technically challenging to determine due to biases arising from the different profiling methods and from striking divergences in the secretory pattern among cellular models [ 18 – 21 ]. The aforementioned broad and potent regulatory function of miRNAs has attracted much attention in recent years, to the point where they are now commonly viewed as major effectors of the intercellular communication function mediated by EVs. EVs can be subcategorized in three main groups based on their size and origin. Exosomes are 50–200 nm in diameter and have the most complex biogenesis, which starts with the formation of endosomal invaginations, thereby creating multivesicular bodies (MVB) (Figure 1). They later fuse to the plasma membrane for the release of exosomes to the extracellular space. This highly intricated and regulated process implies the coordinated action of multiple proteins of the endosomal sorting complex required for transport (ESCRT), Rab GTPases, cytoskeleton, and lipids such as ceramide [ 22 ]. In contrast, microvesicles (also named ectosomes) are 100–1000 nm in diameter and derive from direct budding of the plasma membrane, in a process that shares some steps with the formation of exosomes [ 22 ] (Figure 1). Apoptotic bodies are large vesicles (up to 5000 nm in diameter) that are released when cells undergo apoptosis [ 23 , 24 ]. This classification is subject to continuous debate as emerging modern technologies, such as asymmetric flow field-flow fractionation (AF4), have recently allowed to distinguish different subclasses of EVs within these major groups such as larger and smaller exosomes [ 13 , 24 , 25 ]. In addition, non-membranous extracellular nanoparticles, such as exomeres and supermeres (~35 and 28 nm in average, respectively), have also been identified in biofluids and cell supernatants, although their origin and Int. J. Mol. Sci. 2024,25, 13488 3 of 23 ability to carry miRNAs is still under debate [ 13 , 14 , 26 , 27 ] (Figure 1). The repertoire of non-membranous extracellular miRNA carriers also includes RNA-binding proteins (RBPs) and lipoproteins [ 11 , 12 ]. Although further investigation is definitely needed to better define these new elements, available data indicate that each of these novel subcategories of EVs, nanoparticles, RBPs, and lipoproteins are enriched in particular subsets of cargoes and perform distinct biological functions [ 11 – 14 , 28 ]. Once released, EVs (and potentially also nanoparticles) can interact with the producer (autocrine communication) or nearby cells (paracrine), or travel through the circulation and interact with distant cells (endocrine). Despite some cases of unspecific interaction/uptake by fusion with the plasma membrane of the recipient cell have been reported, most data support that this is a largely cell-type dependent process, which only occurs if the EV and target cell share the right combination of ligand and receptor in their surfaces [ 23 , 29 ] (Figure 1). This selective interaction can trigger EV internalization into endosomal compartments through various mechanisms. These include endocytosis (either dependent on clathrin, caveolin, or lipid raft-associated flotillins), micropinocytosis, and phagocytosis [ 23 , 29 ]. Once in endosomes, the EV cargo can be recycled, re-secreted, or escape to the cytosol where the miRNAs can be incorporated into the miRNA signaling pathway of the recipient cell, leading to phenotypic changes [23,29,30] (Figure 1). Int. J. Mol. Sci. 2024, 25, x FOR PEER REVIEW 3 of 25 non-membranous extracellular nanoparticles, such as exomeres and supermeres (~35 and 28 nm in average, respectively), have also been identified in biofluids and cell supernatants, although their origin and ability to carry miRNAs is still under debate [13,14,26,27] (Figure 1). The repertoire of non-membranous extracellular miRNA carriers also includes RNA-binding proteins (RBPs) and lipoproteins [11,12]. Although further investigation is definitely needed to better define these new elements, available data indicate that each of these novel subcategories of EVs, nanoparticles, RBPs, and lipoproteins are enriched in particular subsets of cargoes and perform distinct biological functions [11–14,28]. Once released, EVs (and potentially also nanoparticles) can interact with the producer (autocrine communication) or nearby cells (paracrine), or travel through the circulation and interact with distant cells (endocrine). Despite some cases of unspecific interaction/uptake by fusion with the plasma membrane of the recipient cell have been reported, most data support that this is a largely cell-type dependent process, which only occurs if the EV and target cell share the right combination of ligand and receptor in their surfaces [23,29] (Figure 1). This selective interaction can trigger EV internalization into endosomal compartments through various mechanisms. These include endocytosis (either dependent on clathrin, caveolin, or lipid raft-associated flotillins), micropinocytosis, and phagocytosis [23,29]. Once in endosomes, the EV cargo can be recycled, re-secreted, or escape to the cytosol where the miRNAs can be incorporated into the miRNA signaling pathway of the recipient cell, leading to phenotypic changes [23,29,30] (Figure 1). Figure 1. EV-carried miRNAs as new players in intercellular communication. In donor cells, miRNAs are loaded into multivesicular bodies (MVB) for the release of exosomes, or into plasma membrane-derived microvesicles. For exosomes, some short sequences named EXOmotifs promote their sorting into forming exosomes by interacting with RNA-binding proteins (RBP). Exosomes, microvesicles, and apoptotic bodies (not depicted in the figure) are commonly referred as extracellular vesicles (EVs). miRNAs are also secreted through unclear mechanisms in other carriers such as RBPs, lipoproteins, and nanoparticles (exomeres and supermeres). By traveling through the circulation, EVs can reach distant cells, where they are internalized by endocytosis and subsequent incorporation into the recipient’s endosomal system, or to a much lesser extent, through direct membrane fusion. In both cases, miRNAs are able to reach the cytosol of the recipient cell and are incorporated into the miRNA signaling pathway by binding to the RISC complex, ultimately leading to the regulation of the expression of mRNA targets in the recipient cell. Figure 1. EV-carried miRNAs as new players in intercellular communication. In donor cells, miRNAs are loaded into multivesicular bodies (MVB) for the release of exosomes, or into plasma membranederived microvesicles. For exosomes, some short sequences named EXOmotifs promote their sorting into forming exosomes by interacting with RNA-binding proteins (RBP). Exosomes, microvesicles, and apoptotic bodies (not depicted in the figure) are commonly referred as extracellular vesicles (EVs). miRNAs are also secreted through unclear mechanisms in other carriers such as RBPs, lipoproteins, and nanoparticles (exomeres and supermeres). By traveling through the circulation, EVs can reach distant cells, where they are internalized by endocytosis and subsequent incorporation into the recipient’s endosomal system, or to a much lesser extent, through direct membrane fusion. In both cases, miRNAs are able to reach the cytosol of the recipient cell and are incorporated into the miRNA signaling pathway by binding to the RISC complex, ultimately leading to the regulation of the expression of mRNA targets in the recipient cell. Int. J. Mol. Sci. 2024,25, 13488 4 of 23 None of the current methods for EV isolation including ultracentrifugation, sizeexclusion chromatography (SEC), or density-gradient centrifugation are able to efficiently separate exosomes from the smallest microvesicles due to their overlapping size [ 24 , 25 , 31 ]. Furthermore, there are no specific markers for each class of EV, as even classical exosomal markers such as tetraspanins CD9, CD63, and CD81 are also present in microvesicles [ 23 , 31 , 32 ], thus preventing their selective isolation by using antibody-based immunoaffinity capture. As a consequence of these technical limitations, there are no efficient methodological approaches that provide a reliable isolation and characterization of the individual categories of EVs. For this reason, and although some publications refer to exosomes or microvesicles as the predominant extracellular particle present in their isolates, we will refer to the general term EVs throughout this review. 3. Adipose Tissue: Extraordinary Secretory Capacity Although extracellular miRNAs are potentially involved in the dialog among all organs in our body, they seem to be particularly important for adipose tissue-based communication. This idea is supported by our initial observation that the deletion of Dicer in adipocytes (ADicerKO mice) is associated with a significant downregulation of nearly two-thirds of the circulating EV-carried miRNAs [ 33 ]. Similar results were obtained in human immunodeficiency virus-1 (VIH-1)-infected patients who developed lipodystrophy, a common comorbidity associated with this pathogen [ 34 , 35 ]. Adipose tissue undergoing lipodystrophy upon HIV-1 infection shows reduced Dicer expression, especially in the dorsocervical region, through unclear mechanisms [ 36 , 37 ]. Although to a lesser extent than in ADicerKO animals, lipodystrophy HIV-1 patients also display a general downregulation in the expression of many circulating EV-carried miRNAs [ 34 ]. These data suggest that a large proportion of circulating EV-carried miRNAs are derived from adipose tissue. Other studies from multiple laboratories have confirmed the extraordinary capacity of adipocytes to secrete EVs, which increases even further in obesity [ 18 , 38 – 41 ]. Although there is no experimental evidence in vivo yet, a plausible explanation is that these extra circulating EVs in obese patients and mice may predominantly derive from expanded adipose tissue, although it is important to note that other tissues also respond to obesity by changing their EV secretion rate and cargo content [ 42 , 43 ]. Despite the large body of evidence supporting the extraordinary capacity of adipocytes to secrete EVs, the molecular mechanisms underlying this phenomenon remain unknown. Similarly, the exact molecular pathways responsible for the different miRNA assortments of adipose tissue-derived EVs (AdEVs) in pathological conditions such as obesity have barely been investigated. One such process could be obesity-induced low-grade chronic inflammation, although many other pathways that are dysregulated in obesity could also play a role [44,45]. How miRNAs (and other cargoes) end up in EVs represents a fascinating new area of research. The seminal studies that first observed bioactive miRNAs in EVs already noted that certain subsets of miRNAs were enriched or depleted in these vesicles compared to their parental cells [ 9 , 10 ], suggesting the existence of miRNA selection mechanisms. This observation has been further confirmed by a long list of subsequent reports [ 46 – 54 ]. Accumulative research has provided important insights into the molecular mechanisms that determine which miRNAs are sorted in EVs and thus may serve a messenger function. Some tetranucleotide sequences (commonly referred to as EXOmotifs) enriched in the miRNAs that tend to be sorted into vesicles were initially described in some particular cell types [ 49 , 51 , 53 ], although other motif-independent mechanisms have been also described [ 48 , 54 ]. Our recent multicellular comparative analysis of EV and cellular miRNAs revealed that each cell type including adipocytes uses a particular subset of EXOmotifs to sort miRNAs in EVs [ 18 ]. While some EXOmotifs are widely present in the EV-enriched miRNAs released by multiple cell types, others are largely cell-type specific. Interestingly, the miRNAs that are selectively depleted from EVs—and therefore enriched in the cellular body—also contain small sequence motifs that we named CELLmotifs. Similar to EXOmotifs, CELLmotifs can be either broadly distributed across different cell types or Int. J. Mol. Sci. 2024,25, 13488 5 of 23 cell-type specific. To further complicate the process of miRNA selection or exclusion from EVs, we identified cases of motifs that behave as an EXOmotif in one cell type and as a CELLmotif in another [ 18 ]. Strong experimental support for EXOmotifs and CELLmotifs to regulate miRNA distribution comes from the fact that adding or removing these motifs from a given miRNA substantially changes its EV versus cellular destination [ 18 , 49 , 51 , 53 ]. The mechanisms guiding the selection of EXOmotif-containing miRNAs or the exclusion of those with CELLmotifs from EVs are far from being understood. Available data suggest that these processes largely rely on RBPs that act selectively in different EXOmotifs and cell types (Figure 1). For instance, in brown adipocytes, ALYREF and FUS participate in the EV loading of CGGGAG-containing miRNAs, the strongest EXOmotif found in this cell type [ 18 ]. Other EXOmotif-reader RBPs include SYNCRIP, hnRNPA2/B1, and Lupus La [ 49 , 51 – 53 ]. However, information on the identity of each motif-reader RBP is lacking for the vast majority of identified EXO and CELLmotifs. Similarly, it still remains unclear how the RBP binds to the miRNA and drives it into the forming EV. The fact that these miRNA-binding RBPs are generally not found within the EV cargo [ 50 ] point that they may allocate the miRNA in the forming EV while they remain in the cell. Interestingly, recent data have shown that the processes of miRNA sorting as well as the EV release rate are regulated by metabolic factors such as insulin or glucagon [ 55 , 56 ]. This raises the exciting possibility that the dysregulation of miRNA sorting mechanisms may account for the altered EV-miRNA signature and subsequent metabolic abnormalities observed in several metabolic diseases such as obesity and diabetes. 4. Adipose Tissue EV Axes 4.1. Technical Limitations for EV Study For adipose tissue, accumulating evidence supports its role as a hub for the release of circulating miRNAs impacting the function of a large number of tissues and cell types. Here, we highlight the most recent findings on AdEV miRNAs that have been shown to shape the metabolism and function of other tissues. As discussed below, investigation over the past decade has identified numerous miRNAs released by adipocytes and other adipose-resident cells that have an impact on the function of another cell type in a distant organ. However, the study of intercellular communication is a technically challenging area of research: the unequivocal demonstration of the adipose origin of a given miRNA is definitely not trivial, as is the validation of its delivery to a particular target cell type due to several limitations associated with current methodologies (Table 1). Therefore, the use of complementary methodologies are recommended to fully demonstrate an interorgan communication role for a given circulating miRNA. One of the procedures often used to identify a potential target tissue is the exogenous administration of EVs, usually after labelling with luminescent, fluorescent, radioactive, or superparamagnetic tracers [ 57 , 58 ] (Table 1). However, caution must be taken before drawing conclusions from this type of experiment. Exogenously-administered EVs (usually at supraphysiologic concentrations) display a marked tropism for the liver, spleen, lungs, and kidneys, all of which are enriched in phagocytes that seem to perform superior EV uptake [ 57 ]. Artificial carriers such as lipid nanoparticles (LNPs) are also preferentially taken up from the circulation by liver or spleen cells, which has favored the development of drug delivery strategies using LNPs to target these tissues [ 59 , 60 ]. In exogenous EV administration experiments, the cellular source for the EVs seems to have a minimal influence in defining the target tissues [ 57 , 61 ]. In contrast, when the EVs are released endogenously, their tissue distribution differs significantly from that of exogenous EVs, with other tissues such as adipose, lung, bone marrow, and gastrointestinal tract leading the EV uptake while liver and spleen were among the tissues with the lowest EV accumulation [ 57 , 62 ]. This suggests that the liver and the other organs where EVs tend to accumulate (lungs, kidneys, and spleen) [ 57 ] might act as sink tissues for supraphysiological doses of exogenous EVs and reinforces the need for robust experimental validation to identify interorgan communication axes in vivo (Table 1). Int. J. Mol. Sci. 2024,25, 13488 6 of 23 Table 1. Current methodologies to study miRNA-based interorgan communication. Table describing the list of common methodologies including their rationality and limitations used to ascribe adipocyte origin to circulating miRNAs as well as for the validation of miRNA delivery to target tissues. Procedures to validate adipose origin of a circulating miRNA Method Rationality Limitations In vitro cell lines miRNAs released to culture medium from adipocyte models 3T3-L1, F442A, PAZ6, etc. Cell lines might not fully recapitulate miRNA secretion pattern from endogenous adipocytes In vitro adipocytes derived from ADSCs miRNAs released to culture medium by adipocytes differentiated in vitro from ADSCs miRNA secretion pattern might be influenced by differentiation cocktail ingredients and their concentrations, efficiency of differentiation and timing Obesity If a circulating miRNA increases in parallel to the increase in fat mass, it might derive from adipocytes Changes in circulating miRNAs might derive from any of the many cell types affected by obesity Adipose markers in EV Capture adipocyte-specific EVs using surface proteins for downstream miRNA profiling Lack of adipose-specific EV marker, although some are enriched (FABP4, perilipin, TGFBI, Adiponectin) Adipose tissue explants or primary adipocytes Incubate fat explants/adipocytes ex vivo and study released miRNAs to the medium Collagenase digestion might disrupt cell membranes and induce the release of cellular miRNA content Incubation in harsh conditions (often serum starved for 24 h) differ from endogenous conditions, potentially inducing cell death and altering miRNA release pattern Explants: released miRNAs could derive from any of the cell types. Isolated adipocytes: lack of extracellular matrix and cell interactions. Floating conditions can alter released miRNA signature Viral vector with adipose-specific promoter An adipocyte promoter (Adiponectin or Fabp4) drives the expression of the miRNA/antagomiR for which the release is being analyzed Some adipocyte promoters might not be fully specific, e.g., Fabp4 also expressed in macrophages and endothelial cells Lipectomy/BATectomy or fat transplantation Removal/transplant of fat from another animal leading to reduced/augmented released levels of a given miRNA These severe procedures might affect released miRNA signatures from fat and other tissues. Other cell types different from adipocytes might be the predominant miRNA source Adypocyte-specific miRNA KO, or Adipocyte-Dicer KO (ADicerKO) mice Deletion of a miRNA gene or Dicer from adipocytes leading to absence/reduction release of that miRNA Good support for adipocyte origin, but it is costly and time consuming. In animals KO for a miRNA, care must be taken to avoid deleting overlapping genes ADicerKO mouse displays a global phenotype, potentially affecting miRNA expression and secretion from other tissues Procedures to validate miRNA delivery to a given tissue/cell type Method Rationality Limitations In vitro cell lines miRNA incorporation upon treatment to cell models in culture Cell lines might not fully recapitulate miRNA uptake behaviour of endogenous cells Administration of labelled EV (e.g., by lipophilic dyes) into mice Detection of the tracer signal in a target tissue The traffic of exogenous EVs potentially differ from endogenous EVs. Lipophilic dyes can form aggregates similar in size to EVs. Viral vector with adipose-specific promoter driving the expression of a EV-localized label Detection of the label signal in another tissue different from adipose Good support for adipose-to-target tissue EV traffic, though does not proof transfer of a specific miRNA Viral vector with adipose-specific promoter driving miRNA mimic/antagomiR expression Detection of enhanced/reduced mature miRNA presence in a tissue different from adipose without changes in precursor miRNA Good support for adipose-to-target tissue miRNA transfer Genetic overexpression of a miRNA or its inhibitor in adipose tissue Detection of enhanced/reduced mature miRNA presence in a tissue different from adipose without changes in precursor miRNA Good support for adipose-to-target tissue miRNA transfer 4.2. Adipose Tissue to Liver Intercellular communication between the liver and adipose tissue has been extensively investigated in recent years. Solid accumulative data support that a large number of EVcarried miRNAs reach the liver cells, where they mediate an important regulatory function over hepatic and systemic metabolism (Figure 2). One of the first studies to demonstrate adipose-to-liver communication via extracellular miRNAs was our work with mice deficient Int. J. Mol. Sci. 2024,25, 13488 7 of 23 for Dicer in adipocytes (ADicerKO) [ 33 ]. These mice developed a massive downregulation of circulating EV miRNAs accompanied by elevated hepatic fibroblast growth factor-21 (Fgf21) mRNA and protein levels. Further experiments confirmed that miR-99b released in brown and white adipocyte EVs can target FGF21 in hepatocytes, thereby regulating the hepatic and circulating levels of this key hormone involved in carbohydrate and lipid metabolism [ 33 , 63 ]. Later studies have reported other white adipocyte-derived miRNAs that control systemic glucose metabolism by targeting the liver. This is the case for miR548ag, whose expression is increased in serum and adipose tissue in obesity. In hepatocytes, this miRNA directly targets the DNA-methyltransferase DNMT3B, which subsequently leads to the upregulation of DPP4, a protein involved in glucose metabolism by degrading incretins such as glucagon-like peptide-1 (GLP-1) [ 64 , 65 ]. Accordingly, the circulating levels of miR-548ag carried by EVs were associated with worsened glucose tolerance and insulin sensitivity [ 64 ]. Similar findings have been described for miR-222, which is also elevated in obesity [ 66 , 67 ]. Gonadal WAT was reported to be the major source of EV-carried miR-222, as removal of this fat depot blunted the high-fat diet (HFD)-induced miR-222 increase in the circulation [ 67 ]. Mechanistically, miR-222 impairs insulin signaling in hepatocytes through targeting IRS1 [ 67 ]. Similarly, miR-4431 is another obesity-associated miRNA that has been linked to worsened glucose tolerance and insulin sensitivity [ 68 ]. Although the expression of this miRNA increases several-fold in the WAT and serum of obese individuals, it reaches even higher levels in the liver, indicating that miR-4431 might also be produced directly by hepatocytes [ 68 ]. In contrast to previous miRNAs that were upregulated in obesity, miR-141-3p was found to be downregulated in adipose-derived EVs isolated from geneticand diet-induced obesity models. By targeting phosphatase and tensin homolog (PTEN), this miRNA was shown to inhibit insulin sensitivity and glucose uptake in hepatocytes in vitro (AML12 cells) [69]. Int. J. Mol. Sci. 2024, 25, x FOR PEER REVIEW 8 of 25 This is the case for miR-548ag, whose expression is increased in serum and adipose tissue in obesity. In hepatocytes, this miRNA directly targets the DNA-methyltransferase DNMT3B, which subsequently leads to the upregulation of DPP4, a protein involved in glucose metabolism by degrading incretins such as glucagon-like peptide-1 (GLP-1) [64,65]. Accordingly, the circulating levels of miR-548ag carried by EVs were associated with worsened glucose tolerance and insulin sensitivity [64]. Similar findings have been described for miR-222, which is also elevated in obesity [66,67]. Gonadal WAT was reported to be the major source of EV-carried miR-222, as removal of this fat depot blunted the high-fat diet (HFD)-induced miR-222 increase in the circulation [67]. Mechanistically, miR-222 impairs insulin signaling in hepatocytes through targeting IRS1 [67]. Similarly, miR-4431 is another obesity-associated miRNA that has been linked to worsened glucose tolerance and insulin sensitivity [68]. Although the expression of this miRNA increases several-fold in the WAT and serum of obese individuals, it reaches even higher levels in the liver, indicating that miR-4431 might also be produced directly by hepatocytes [68]. In contrast to previous miRNAs that were upregulated in obesity, miR-141-3p was found to be downregulated in adipose-derived EVs isolated from geneticand diet-induced obesity models. By targeting phosphatase and tensin homolog (PTEN), this miRNA was shown to inhibit insulin sensitivity and glucose uptake in hepatocytes in vitro (AML12 cells) [69]. Figure 2. Adipose-derived miRNAs delivered to other metabolic organs. Scheme representing the miRNAs released by WAT/BAT and delivered to the skeletal muscle, liver, pancreatic islets, brain, cardiovascular system, and gut. The numbers in brackets indicate the reference in the main text. Aside from white adipocytes, brown adipocytes are also important senders of AdEVloaded miRNAs targeting the liver. Indeed, despite their lower mass, brown adipocytes appear to be stronger EV sender cells compared to white adipocytes. This was evidenced by the superior restoration of circulating miRNA levels in ADicerKO mice after the transplantation of wild-type brown adipose tissue (BAT) compared to a lower, but still remarkable, restoration when transplanted white adipose tissue (WAT) [33]. The functional consequences of the BAT-to-liver axis have been further illustrated in cold-exposed mice. In these conditions, brown adipocytes release more EVs, which contain, among others, miR378a-3p and miR-132-3p. These two miRNAs reach hepatocytes, where they promote gluconeogenesis by targeting p110α (by miR-378a-3p) and repress lipogenesis by SREBF1 targeting (by miR-132-3p) [70,71]. Figure 2. Adipose-derived miRNAs delivered to other metabolic organs. Scheme representing the miRNAs released by WAT/BAT and delivered to the skeletal muscle, liver, pancreatic islets, brain, cardiovascular system, and gut. The numbers in brackets indicate the reference in the main text. Aside from white adipocytes, brown adipocytes are also important senders of AdEVloaded miRNAs targeting the liver. Indeed, despite their lower mass, brown adipocytes appear to be stronger EV sender cells compared to white adipocytes. This was evidenced by the superior restoration of circulating miRNA levels in ADicerKO mice after the transplantation of wild-type brown adipose tissue (BAT) compared to a lower, but still remarkable, Int. J. Mol. Sci. 2024,25, 13488 8 of 23 restoration when transplanted white adipose tissue (WAT) [ 33 ]. The functional consequences of the BAT-to-liver axis have been further illustrated in cold-exposed mice. In these conditions, brown adipocytes release more EVs, which contain, among others, miR-378a-3p and miR-132-3p. These two miRNAs reach hepatocytes, where they promote gluconeogenesis by targeting p110 α (by miR-378a-3p) and repress lipogenesis by SREBF1 targeting (by miR-132-3p) [70,71]. In addition to mature adipocytes, other adipose-resident cells release miRNAs that have important implications for hepatic, and hence, systemic metabolism. A good example is adipose tissue macrophages, whose EV-carried miRNA profile significantly differs in obese versus lean conditions. Some of these miRNAs, such as miR-155 and miR-690, correlate positively or negatively, respectively, with insulin resistance in hepatocytes, adipocytes, and muscle cells [ 72 , 73 ]. Other adipose-resident cells that have been extensively studied in this context are adipose-derived stem cells (ADSCs) due to their major regenerative properties in different tissues and conditions [ 74 ], as further outlined in the next sections. Their released EVs can reach the liver, among other tissues, where they exert regenerative functions. For instance, ADSC-released miR-223-3p suppresses hepatic lipid accumulation and fibrosis by E2F1 targeting, while miR-144-3p and miR-486a-3p activate hepatocyte proliferative pathways by suppressing TXNIP expression [75,76]. Reciprocal communication from the liver to adipose tissue mediated by EVs has been much less studied than the reverse direction. In this regard, some reports have shown that hepatic EVs promote glucose uptake in adipocytes through a variety of mechanisms, one of which is the miR-130a-3p-mediated targeting of PHLPP2, a modulator of the AKT-GLUT4 axis [ 77 , 78 ]. Adipocyte targeting of hepatic EVs also plays a role in disease such as obesity and fatty liver. Specifically, obesity implies changes in the miRNA content of EVs released from primary hepatocytes. A subset of these miRNAs, with the strongest effect mediated by let-7e-5p, has been shown to promote lipid accumulation in adipocytes [ 43 ]. Some of these miRNAs were found to be elevated in circulating EVs from patients with concomitant obesity and fatty liver but not in those with a fatty liver alone, suggesting a potential involvement of these hepatic miRNAs in fat mass gain [43]. 4.3. Adipose Tissue to Skeletal Muscle Overall, the regulatory potential of AdEV-carried miRNAs in skeletal muscle metabolism and function has been less studied than in the liver, but still, a good number of miRNAs have been shown to be involved in this communication axis (Figure 2). One of the first miRNAs reported to be involved in adipocyte-to-muscle communication was miR-130b [ 79 ]. This miRNA was found to inhibit the skeletal muscle expression of PGC-1 α , a master regulator of lipid oxidation and mitochondrial biogenesis [ 79 ]. Later on, miR-27a, whose levels increase in adipose tissue, serum, and skeletal muscle under obese conditions, was shown to promote muscle insulin resistance through the inhibition of PPAR γ , at least in an in vitro myotube model [ 80 ]. Similarly, obesity-associated circulating miR-222 [ 66 ], in addition to hepatocytes, can also reach muscle cells to downregulate IRS1 and insulin signaling, thereby contributing to insulin resistance and glucose intolerance in obesity [ 67 ]. Indeed, obesity also alters the secretion and cargo content of EVs secreted by muscle cells. Interestingly, in contrast to the enhanced secretion of EVs by adipose tissue in obesity [ 40 , 41 ], muscle explants from obese animals secrete a lower number of EVs compared to lean explants [ 42 ]. The miRNAs contained in these EVs may also be key factors contributing to boost obesityinduced fat gain, as they have been shown to promote lipid accumulation in adipocytes [ 42 ]. Beyond the obesity context, there are other situations that illustrate the close reciprocal crosstalk between muscle and adipose tissue mediated by EV-miRNAs. For instance, promoting muscle activity through exercise or synergist ablation results in the enhanced delivery of EV-loaded miR-1 to adipocytes, thereby enhancing catecholamine-induced lipolysis [81,82]. EV-carried miRNAs have also emerged as key elements for the crosstalk between muscle and adipose progenitors. For instance, adipocyte progenitors residing in skeletal Int. J. Mol. Sci. 2024,25, 13488 9 of 23 muscle (fibroadipogenic progenitors, FAPs) participate in muscle regeneration upon injury. FAPs secrete EV-carried miRNAs (such as miR-127-3p) in a paracrine manner to release muscle stem cells (MuSCs) from quiescence by targeting the myogenic gene S1PR3, thus allowing muscle mass recovery [ 83 , 84 ]. Conversely, MuSCs can also have an influence on adipose progenitors. For instance, MuSCs can reduce the proliferation and differentiation of ADSCs by secreting EV-loaded miR-146-5p, a miRNA previously shown to target insulin receptor [85,86]. 4.4. Adipose Tissue to Pancreatic Islets Despite the pivotal role of pancreatic islets in the regulation of systemic metabolism, the study of the potential contribution of adipocyte-released miRNAs to the physiology and pathology of beta cells and other islet cells has barely been investigated in comparison to other organs. However, some recent reports have identified subsets of circulating miRNAs with potential adipocyte origin that regulate beta cell proliferation and activity (Figure 2). For instance, miR-132-3p, whose expression is elevated in visceral adipocytes and pancreatic islets under obese conditions, is linked to enhanced beta cell regeneration [ 87 – 90 ]. Similarly, miR-15b and miR-146b suppressed insulin secretion in an in vitro model of beta cells [ 91 ]. However, whether adipocytes represent the main source of the circulating pool of these three aforementioned miRNAs was not verified [ 91 ]. In contrast, the adipocyte origin was confirmed for another cluster of EV-carried miRNAs (miR-29a-3p, miR-200a-3p, miR-218-5p and miR-322-5p) that inhibit early insulin secretion by primary islets and a beta cell line (MIN6) exposed to high glucose conditions [ 92 ]. These miRNAs were identified by looking at the EVs released by epidydimal obese adipocytes into cell culture medium [ 92 ]. Using an alternative approach, Zhang and colleagues inferred miR-27a-5p as an adipocyte EV-carried miRNA regulating insulin secretion in beta cells [ 93 ]. In this case, miR-27a-5p levels increase in mouse and human islets under obesity conditions while its precursor decreases, suggesting an extra-islet origin. The rise in the expression of both mature and precursor miR-27a with obesity specifically in epididymal fat suggested an adipocyte origin for this miRNA. Further in vivo experiments using adipocyte-specific overexpression or neutralization approaches confirmed the EV-mediated transport of this miRNA from adipocytes to islets, where it targeted a calcium transporter (CACNA1C) and thereby blunted insulin secretion [ 93 ]. The initial trigger for the elevated secretion of these previous miRNAs in EVs from adipocytes subjected to obesity conditions was not ascertained. In this regard, AdEVs released by adipocytes that were previously exposed to inflammatory cytokines have been shown to promote beta cell apoptosis and impair insulin secretion in recipient beta cells in vitro [ 44 ]. These deleterious effects may be mediated, at least in part, by the distinct AdEV-miRNA signature induced by the pretreatment with proinflammatory factors [ 44 ]. Taken together, these data suggest that miRNAs released from inflamed adipocytes may be involved in the metabolic deterioration of beta cells in obesity and diabetes. Aside from adipocytes, comprehensive in vivo experiments have also confirmed the release of proinflammatory miR-155-5p in adipose tissue macrophagederived EVs, which are able to suppress insulin secretion and cellular proliferation in beta cells in vivo [ 94 ]. The potential regulatory actions of miRNAs released from adipose tissue cells on other pancreatic islet cells different from beta cells remain to be investigated. 4.5. Adipose Tissue to Cardiovascular System Fat accumulation, especially in visceral depots, is strongly associated with adverse cardiovascular events [ 95 ]. The contributing role of adipose tissue in cardiovascular disease (CVD) is illustrated by the protective effect of visceral fat removal in several mouse models of cardiac dysfunction [ 96 , 97 ]. Due to its anatomical proximity to the myocardium, epicardial adipose tissue (EAT), a subtype of visceral fat depot, has received much attention as a potential source of AdEV-carried miRNAs regulating cardiac function. Indeed, several miRNAs released in EAT-derived AdEVs have been shown to regulate metabolism, the production of reactive oxygen species, and contractile function when administered to ven- Int. J. Mol. Sci. 2024,25, 13488 16 of 23 Funding: This work was supported by a grant PID2022-141519OA-I00, funded by Spanish Ministry of Science, Innovation, and Universities/Spanish Agency of Investigation/European Union (MICIU/AEI, ref. 10.13039/501100011033), and by a grant RYC2021-033875-I funded by the Spanish National Research Council (CSIC) to R.G.-M. A Formacion Personal Investigador (PFI) fellowship (PREP2022-000779) supported P.D.-R. We used Biorender for the drawing of the figures displayed in this article. Conflicts of Interest: The authors declare no conflicts of interest. References 1. Ouchi, N.; Parker, J.L.; Lugus, J.J.; Walsh, K. Adipokines in inflammation and metabolic disease. Nat. Rev. Immunol. 2011, 11, 85–97. [CrossRef] 2. 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