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The role of extracellular vesicle-derived miRNAs in adipose tissue function and metabolic health
Abstract
Extracellular vesicles (EVs) are nanometer size lipid particles that are released from virtually every cell type. Recent studies have shown that miRNAs carried by EVs play important roles in intercellular and interorgan communication. In the context of obesity and insulin resistance, EV-derived miRNAs functionally bridge major metabolic organs, including the adipose tissue, skeletal muscle, liver, and pancreas, to regulate insulin secretion and signaling. As a result, many of these EV-derived miRNAs have been proposed as potential disease biomarkers and/or therapeutic agents. However, the field’s knowledge of EV miRNA-mediated regulation of mammalian metabolism is still in its infancy. Here, we review the evidence indicating that EV-derived miRNAs provide cell-to-cell and organ-to-organ communication to support metabolic health, highlight the potential medical relevance of these discoveries, and discuss the most important knowledge gaps and future directions for this field.
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Skeletal muscle-fat interaction is essential for maintaining organismal energy homeostasis and managing obesity by secreting cytokines and exosomes, but the role of the latter as a new mediator in inter-tissue communication remains unclear. Recently, we discovered that miR-146a-5p was mainly enriched in skeletal muscle-derived exosomes (SKM-Exos), 50-fold higher than in fat exosomes. Here, we investigated the role of skeletal muscle-derived exosomes regulating lipid metabolism in adipose tissue by delivering miR-146a-5p. The results showed that skeletal muscle cell-derived exosomes significantly inhibited the differentiation of preadipocytes and their adipogenesis. When the skeletal muscle-derived exosomes co-treated adipocytes with miR-146a-5p inhibitor, this inhibition was reversed. Additionally, skeletal muscle-specific knockout miR-146a-5p (mKO) mice significantly increased body weight gain and decreased oxidative metabolism. On the other hand, the internalization of this miRNA into the mKO mice by injecting skeletal muscle-derived exosomes from the Flox mice (Flox-Exos) resulted in significant phenotypic reversion, including down-regulation of genes and proteins involved in adipogenesis. Mechanistically, miR-146a-5p has also been demonstrated to function as a negative regulator of peroxisome proliferator-activated receptor γ (PPARγ) signaling by directly targeting growth and differentiation factor 5 (GDF5) gene to mediate adipogenesis and fatty acid absorption. Taken together, these data provide new insights into the role of miR-146a-5p as a novel myokine involved in the regulation of adipogenesis and obesity via mediating the skeletal muscle-fat signaling axis, which may serve as a target for the development of therapies against metabolic diseases, such as obesity.
Exosomes and other small extracellular vesicles (sEVs) provide a unique mode of cell-to-cell communication in which microRNAs (miRNAs) produced and released from one cell are taken up by cells at a distance where they can enact changes in gene expression1,2,3. However, the mechanism by which miRNAs are sorted into exosomes/sEVs or retained in cells remains largely unknown. Here we demonstrate that miRNAs possess sorting sequences that determine their secretion in sEVs (EXOmotifs) or cellular retention (CELLmotifs) and that different cell types, including white and brown adipocytes, endothelium, liver and muscle, make preferential use of specific sorting sequences, thus defining the sEV miRNA profile of that cell type. Insertion or deletion of these CELLmotifs or EXOmotifs in a miRNA increases or decreases retention in the cell of production or secretion into exosomes/sEVs. Two RNA-binding proteins, Alyref and Fus, are involved in the export of miRNAs carrying one of the strongest EXOmotifs, CGGGAG. Increased miRNA delivery mediated by EXOmotifs leads to enhanced inhibition of target genes in distant cells. Thus, this miRNA code not only provides important insights that link circulating exosomal miRNAs to tissues of origin, but also provides an approach for improved targeting in RNA-mediated therapies.
Skeletal muscles secrete various factors, such as proteins/peptides, nucleotides, and metabolites, which are referred to as myokines. Many of these factors are transported into extracellular bodily fluids in a free or protein-bound form. Furthermore, several secretory factors have been shown to be wrapped up by small vesicles, particularly exosomes, secreted into circulation, and subsequently regulate recipient cells. Thus, exosome contents can be recognized as myokines. In recipient cells, proteins, microRNAs, and metabolites in exosomes can regulate the expression and activity of target proteins associated with nutrient metabolism and immune function. The levels of circulating exosomes and their contents are altered in muscle disorders and metabolic-related states, such as metabolic dysfunction, sarcopenia, and physical fitness. Therefore, such circulating factors could mediate various interactions between skeletal muscle and other organs and may be useful as biomarkers reflecting physiological and pathological states associated with muscular function. Here, this review summarizes secretory regulation of muscle-derived exosomes. Their metabolic and immunological roles and the significance of their circulating levels are also discussed.
In chronic obesity, hepatocytes become insulin resistant and exert important effects on systemic metabolism. Here we show that in early onset obesity (4 weeks high-fat diet), hepatocytes secrete exosomes that enhance insulin sensitivity both in vitro and in vivo. These beneficial effects were due to exosomal microRNA miR-3075, which is enriched in these hepatocyte exosomes. FA2H is a direct target of miR-3075 and small interfering RNA depletion of FA2H in adipocytes, myocytes and primary hepatocytes leads to increased insulin sensitivity. In chronic obesity (16–18 weeks of a high-fat diet), hepatocyte exosomes promote a state of insulin resistance. These chronic obese hepatocyte exosomes do not directly cause impaired insulin signalling in vitro but do promote proinflammatory activation of macrophages. Taken together, these studies show that in early onset obesity, hepatocytes produce exosomes that express high levels of the insulin-sensitizing miR-3075. In chronic obesity, this compensatory effect is lost and hepatocyte-derived exosomes from chronic obese mice promote insulin resistance.
Exosomes are small extracellular vesicles 40–160 nm in diameter that are secreted by almost all cell types. Exosomes can carry diverse cargo including RNA, DNA, lipids, proteins, and metabolites. Exosomes transfer substances and information between cells by circulating in body fluids and are thus involved in diverse physiological and pathological processes in the human body. Recent studies have closely associated exosomal microRNAs (miRNAs) with various human diseases, including diabetes mellitus (DM), which is a complex multifactorial metabolic disorder disease. Exosomal miRNAs are emerging as pivotal regulators in the progression of DM, mainly in terms of pancreatic β‐cell injury and insulin resistance. Exosomal miRNAs are closely associated with DM‐associated complications, such as diabetic retinopathy (DR), diabetic nephropathy (DN), and diabetic cardiomyopathy (DCM), etc. Further investigations of the mechanisms of action of exosomal miRNAs and their role in DM will be valuable for the thorough understanding of the physiopathological process of DM. Here, we have summarized recent findings regarding exosomal miRNAs associated with DM to provide a new strategy for identifying potential diagnostic biomarkers and drug targets for the early diagnosis and treatment, respectively, of DM. Recent studies have closely associated exosomal microRNAs (miRNAs) with various human diseases, including diabetes mellitus (DM), which is a complex multifactorial metabolic disorder disease. In the diabetic condition, exosomal miRNAs are taken up by recipient cells, where they exert their biological function and thereby modulate the progression of DM‐associated complications, including diabetic retinopathy (DR), diabetic macrovascular complications (DMCs), diabetic nephropathy (DN), diabetic foot ulcer (DFU), diabetic peripheral neuropathy (DPN), and diabetic cardiomyopathy (DCM).
Aims/hypothesis
Macrophage levels are elevated in pancreatic islets, and the resulting inflammatory response is a major contributor to beta cell failure during obesity and type 2 diabetes mellitus. Previous studies by us and others have reported that exosomes released by macrophages play important roles in mediating cell-to-cell communication, and represent a class of inflammatory factors involved in the inflammatory process associated with type 2 diabetes mellitus. However, to date, no reports have demonstrated the effect of macrophage-derived exosomes on beta cells, and little is known regarding their underlying mechanisms in beta cell injury. Thus, we aimed to study the impact of macrophage-derived exosomes on islet beta cell injury in vitro and in vivo.
Methods
The phenotypic profiles of islet-resident macrophages were analysed in C57BL/6J mice fed a high-fat diet (HFD). Exosomes were collected from the medium of cultured bone marrow-derived macrophages (BMDMs) and from isolated islet-resident macrophages of HFD-fed mice (HFD-Exos). The role of exosomes secreted by inflammatory M1 phenotype BMDMs (M1-Exos) and HFD-Exos on beta cell function was assessed. An miRNA microarray and quantitative real-time PCR (qPCR) were conducted to test the level of M1-Exos-derived miR-212-5p in beta cells. Then, miR-212-5p was overexpressed or inhibited in M1-Exos or beta cells to determine its molecular and functional impact.
Results
M1-polarised macrophages were enriched in the islets of obese mice. M1 macrophages and islet-resident macrophages of HFD-fed mice impaired beta cell insulin secretion in an exosome-dependent manner. miR-212-5p was notably upregulated in M1-Exos and HFD-Exos. Enhancing the expression of miR-212-5p impaired beta cell insulin secretion. Blocking miR-212-5p elicited a significant improvement in M1-Exos-mediated beta cell insulin secretion during injury. Mechanistically, M1-Exos mediated an intercellular transfer of the miR-212-5p, targeting the sirtuin 2 gene and regulating the Akt/GSK-3β/β-catenin pathway in recipient beta cells to restrict insulin secretion.
Conclusions/interpretation
A novel exosome-modulated mechanism was delineated for macrophage-beta cell crosstalk that drove beta cell dysfunction and should be explored for its therapeutic utility.
Graphical abstract
Exosomes are small vesicles (30–150 nm in diameter) enclosed by a lipid membrane bilayer, secreted by most cells in the body. They carry various molecules, including proteins, lipids, mRNA, and other RNA species, such as long non-coding RNA, circular RNA, and microRNA (miRNA). miRNAs are the most numerous cargo molecules in the exosome. They are endogenous non-coding RNA molecules, approximately 19–22-nt-long, and important regulators of protein biosynthesis. Exosomes can be taken up by neighboring or distant cells, where they play a role in post-transcriptional regulation of gene expression by targeting mRNA. Exosomal miRNAs have diverse functions, such as participation in inflammatory reactions, cell migration, proliferation, apoptosis, autophagy, and epithelial–mesenchymal transition. There is increasing evidence that exosomal miRNAs play an important role in cardiovascular health. Exosomal miRNAs are widely involved in the occurrence and development of cardiovascular diseases, such as atherosclerosis, acute coronary syndrome, heart failure (HF), myocardial ischemia reperfusion injury, and pulmonary hypertension. In this review, we present a systematic overview of the research progress into the role of exosomal miRNAs in cardiovascular diseases, and present new ideas for the diagnosis and treatment of cardiovascular diseases.
Exosomes are cell-derived nanovesicles that are involved in the intercellular transportation of materials. Therapeutics, such as small molecules or nucleic acid drugs, can be incorporated into exosomes and then delivered to specific types of cells or tissues to realize targeted drug delivery. Targeted delivery increases the local concentration of therapeutics and minimizes side effects. Here, we present a detailed review of exosomes engineering through genetic and chemical methods for targeted drug delivery. Although still in its infancy, exosome-mediated drug delivery boasts low toxicity, low immunogenicity, and high engineerability, and holds promise for cell-free therapies for a wide range of diseases.
Angiogenesis and osteogenesis are tightly coupled during bone modeling and remodeling processes. Here we reported that bone marrow mesenchymal stem cell (BMSC)-derived exosomal miR-29a promotes angiogenesis and osteogenesis in vitro and in vivo. BMSC-derived exosomes (BMSCs-Exos) can be taken up by human umbilical vein endothelial cells (HUVECs) and promote the proliferation, migration, and tube formation of HUVECs. MiRNA-29a level was high in BMSCs-Exos and can be transported into HUVECs to regulate angiogenesis. VASH1 was identified as a direct target of miR-29a, mediating the effects of BMSC-derived exosomal miR-29a on angiogenesis. More interestingly, miR29a-loaded exosomes from engineered BMSCs (miR-29a-loaded BMSCs-Exos) showed a robust ability of promoting angiogenesis and osteogenesis in vivo. Taken together, these findings suggest that BMSC-derived exosomal miR-29a regulates angiogenesis and osteogenesis, and miR-29a-loaded BMSCs-Exos may serve as a potential therapeutic target for osteoporosis.
Objective
To evaluate serum microRNA (miR)-29a/b expression in gestational diabetes mellitus (GDM) and its influence on neonatal prognosis.
Methods
This was a retrospective study including 68 pregnant women with GDM (GDM group) and 55 healthy pregnant women of similar age range and gestation period (healthy group).
Results
The area under the curve was 0.829 for the diagnosis of GDM using serum miR-29a expression, 0.857 for diagnosis using serum miR-29b expression, and 0.944 for combined diagnosis (using both miR-29a and miR-29b). The fasting insulin (FINS) level of the GDM group was significantly lower than that of the healthy group; levels of fasting plasma glucose (FPG), 2-h plasma glucose (2hPG), and glycated hemoglobin (HbA1c) were significantly higher in the GDM group than in the healthy group. Both miR-29a and miR-29b were positively correlated with FINS levels and negatively correlated with FPG, 2hPG, and HbA1c levels. Serum miR-29a/b expression in pregnant women with GDM was not correlated with neonatal weight, premature delivery, or asphyxia but was correlated with pathologic jaundice.
Conclusions
Serum miR-29a/b expression was downregulated in pregnant women with GDM and correlated with neonatal pathologic jaundice, showing good individual (miR-29a or miR-29b) diagnostic value and excellent combined (miR-29a and miR-29b) diagnostic value.
Extracellular vesicles (EVs) are membranous vesicles secreted by both prokaryotic and eukaryotic cells and play a vital role in intercellular communication. EVs are classified into several subtypes based on their origin, physical characteristics, and biomolecular makeup. Exosomes, a subtype of EVs, are released by the fusion of multivesicular bodies (MVB) with the plasma membrane of the cell. Several methods have been described in literature to isolate exosomes from biofluids including blood, urine, milk, and cell culture media, among others. While differential ultracentrifugation (dUC) has been widely used to isolate exosomes, other techniques including ultrafiltration, precipitating agents such as poly-ethylene glycol (PEG), immunoaffinity capture, microfluidics, and size-exclusion chromatography (SEC) have emerged as credible alternatives with pros and cons associated with each. In this review, we provide a summary of commonly used exosomal isolation techniques with a focus on SEC as an ideal methodology. We evaluate the efficacy of SEC to isolate exosomes from an array of biological fluids, with a particular focus on its application to adipose tissue-derived exosomes. We argue that exosomes isolated via SEC are relatively pure and functional, and that this methodology is reproducible, scalable, inexpensive, and does not require specialized equipment or user expertise. However, it must be noted that while SEC is a good candidate method to isolate exosomes, direct comparative studies are required to support this conclusion.
We report a covalent chemistry-based hepatocellular carcinoma (HCC)-specific extracellular vesicle (EV) purification system for early detection of HCC by performing digital scoring on the purified EVs. Earlier detection of HCC creates more opportunities for curative therapeutic interventions. EVs are present in circulation at relatively early stages of disease, providing potential opportunities for HCC early detection. We develop an HCC EV purification system (i.e., EV Click Chips) by synergistically integrating covalent chemistry-mediated EV capture/ release, multimarker antibody cocktails, nanostructured substrates, and microfluidic chaotic mixers. We then explore the translational potential of EV Click Chips using 158 plasma samples of HCC patients and control cohorts. The purified HCC EVs are subjected to reverse-transcription droplet digital PCR for quantification of 10 HCC-specific mRNA markers and computation of digital scoring. The HCC EV-derived molecular signatures exhibit great potential for noninvasive early detection of HCC from at-risk cirrhotic patients with an area under receiver operator characteristic curve of 0.93 (95% CI, 0.86 to 1.00; sensitivity = 94.4%, specificity = 88.5%).
Neurodegenerative diseases (NDDs) are a group of diseases caused by chronic and progressive degeneration of neural tissue. The main pathological manifestations are neuronal degeneration and loss in the brain and/or spinal cord. Common NDDs include Alzheimer disease (AD), Parkinson disease (PD), Huntington disease (HD), and amyotrophic lateral sclerosis (ALS). The complicated pathological characteristics and different clinical manifestations of NDDs result in a lack of sensitive and efficient diagnostic methods. In addition, no sensitive biomarkers are available to monitor the course of NDDs, predict their prognosis, and monitor the therapeutic response. Despite extensive research in recent years, analysis of amyloid β (Aβ) and α-synuclein has failed to effectively improve NDD diagnosis. Although recent studies have indicated circulating miRNAs as promising diagnostic biomarkers of NDDs, the miRNA in the peripheral circulation is susceptible to interference by other components, making circulating miRNA results less consistent. Exosomes are small membrane vesicles with a diameter of approximately 30–100 nm that transport proteins, lipids, mRNA, and miRNA. Because recent studies have shown that exosomes have a double-membrane structure that can resist ribonuclease in the blood, giving exosomal miRNA high stability and making them resistant to degradation, they may become an ideal biomarker of circulating fluids. In this review, we discuss the applicability of circulating exosomal miRNAs as biomarkers, highlight the technical aspects of exosomal miRNA analysis, and review studies that have used circulating exosomal miRNAs as candidate diagnostic biomarkers of NDDs.
Objective:
Type 2 diabetes mellitus (T2DM) is featured by insulin resistance and lipid metabolism dysregulation. A large number of miRNAs were identified in exosomes derived from adipose tissue macrophages associated with T2DM pathogenesis, but its pathogenic roles remain unknown. This study is aimed at investigating the function of miR-210 in diabetic obesity.
Methods:
Exosomes from mouse macrophage RAW264.7 cells were characterized by electron microscopy, combined with biomarker expression by western blot. Expression of miR-210 was determined by quantitative RT-PCR. Glucose uptake was measured by a fluorometric method, and the mitochondrial respiratory chain activity was evaluated by ELISA. The target gene of miR-210 was validated by dual-luciferase reporter and pull-down assays. A mouse obese diabetic model was established by a high-fat diet and streptozocin treatment.
Results:
miR-210 was highly expressed in exosomes derived from high glucose-induced macrophage RAW264.7 cells. Macrophage-derived exosomes impaired glucose uptake and mitochondrial CIV complex activity and suppressed NADH dehydrogenase ubiquinone 1 alpha subcomplex 4 (NDUFA4) expression in 3T3-L1 adipocytes. miR-210 directly bind with mRNA sequences of NDUFA4 gene. Inhibition of miR-210 mitigated the effects of macrophage-derived exosomes on the glucose uptake and complex IV (CIV) activity in 3T3-L1 adipocytes, and NDUFA4 overexpression offset the inhibition of glucose uptake and CIV activity by macrophage-derived exosomes. Furthermore, mice with miR-210 knockout showed greatly repressed diabetic obesity development.
Conclusion:
miR-210 derived from adipose tissue macrophages promotes mouse obese diabetes pathogenesis by regulating glucose uptake and mitochondrial CIV activity through targeting NDUFA4 gene expression.
Type 2 diabetes (T2D) is characterized by insulin resistance along with pancreatic β cell failure. β cell factors are traditionally thought to control glucose homeostasis by modulating insulin levels, not insulin sensitivity. Exosomes are emerging as new regulators of intercellular communication. However, the role of β-cell–derived exosomes in metabolic homeostasis is poorly understood. Here, we report that microRNA-26a (miR-26a) in β cells not only modulates insulin secretion and β cell replication in an autocrine manner but also regulates peripheral insulin sensitivity in a paracrine manner through circulating exosomes. MiR-26a is reduced in serum exosomes of overweight humans and is inversely correlated with clinical features of T2D. Moreover, miR-26a is down-regulated in serum exosomes and islets of obese mice. Using miR-26a knockin and knockout mouse models, we showed that miR-26a in β cells alleviates obesity-induced insulin resistance and hyperinsulinemia. Mechanistically, miR-26a in β cells enhances peripheral insulin sensitivity via exosomes. Meanwhile, miR-26a prevents hyperinsulinemia through targeting several critical regulators of insulin secretion and β cell proliferation. These findings provide a new paradigm for the far-reaching systemic functions of β cells and offer opportunities for the treatment of T2D.
An increasing number of studies have reported that exosomes released from various cells can serve as mediators of information exchange between different cells. With further exploration of exosome content, a more accurate molecular mechanism involved in the process of cell-to-cell communication has been revealed; specifically, microRNAs (miRNAs) and long noncoding RNAs (lncRNAs) are shuttled by exosomes. In addition, exosomal miRNAs and lncRNAs may play vital roles in the pathogenesis of several respiratory diseases, such as chronic obstructive pulmonary disease (COPD), lung cancer, and asthma. Consequently, exosomal miRNAs and lncRNAs show promise as diagnostic biomarkers and therapeutic targets in several lung diseases. This review will summarize recent knowledge about the roles of exosomal miRNAs and lncRNAs in lung diseases, which has shed light on the discovery of novel diagnostic methods and treatments for these disorders. Because there is almost no published literature about exosomal lncRNAs in COPD, asthma, interstitial lung disease, or tuberculosis, we summarize the roles of exosomal lncRNAs only in lung cancer in the second section. This may inspire some new ideas for researchers who are interested in whether lncRNAs shuttled by exosomes may play roles in other lung diseases.
Extracellular vesicles (EVs) encompass a variety of vesicles secreted into the extracellular space. EVs have been implicated in promoting tumor metastasis, but the molecular composition of tumor-derived EV sub-types and the mechanisms by which molecules are sorted into EVs remain mostly unknown. We report the separation of two small EV sub-populations from a metastatic breast cancer cell line, with biochemical features consistent with different sub-cellular origins. These EV sub-types use different mechanisms of miRNA sorting (selective and non-selective), suggesting that sorting occurs via fundamentally distinct processes, possibly dependent on EV origin. Using biochemical and genetic tools, we identified the Lupus La protein as mediating sorting of selectively packaged miRNAs. We found that two motifs embedded in miR-122 are responsible for high-affinity binding to Lupus La and sorting into vesicles formed in a cell-free reaction. Thus, tumor cells can simultaneously deploy multiple EV species using distinct sorting mechanisms that may enable diverse functions in normal and cancer biology.
Obesity is a critical risk factor for the development of type 2 diabetes (T2D), and its prevalence is rising worldwide. White adipose tissue (WAT) has a crucial role in regulating systemic energy homeostasis. Adipose tissue expands by a combination of an increase in adipocyte size (hypertrophy) and number (hyperplasia). The recruitment and differentiation of adipose precursor cells in the subcutaneous adipose tissue (SAT), rather than merely inflating the cells, would be protective from the obesity-associated metabolic complications. In metabolically unhealthy obesity, the storage capacity of SAT, the largest WAT depot, is limited, and further caloric overload leads to the fat accumulation in ectopic tissues (e.g., liver, skeletal muscle, and heart) and in the visceral adipose depots, an event commonly defined as “lipotoxicity.” Excessive ectopic lipid accumulation leads to local inflammation and insulin resistance (IR). Indeed, overnutrition triggers uncontrolled inflammatory responses in WAT, leading to chronic low-grade inflammation, therefore fostering the progression of IR. This review summarizes the current knowledge on WAT dysfunction in obesity and its associated metabolic abnormalities, such as IR. A better understanding of the mechanisms regulating adipose tissue expansion in obesity is required for the development of future therapeutic approaches in obesity-associated metabolic complications.
Advanced diabetes mellitus (DM) may have both insulin resistance and deficiency (double DM) that accelerates diabetic cardiomyopathy (DMCM), a cardiac muscle disorder. Reduced cardiac miR-133a, a cardioprotective miRNA, is associated with DMCM. However, it is unclear whether increasing miR-133a levels in the double DM heart could prevent DMCM. We hypothesized that increasing cardiac levels of miR-133a could prevent DMCM in Akita, a mouse model of double DM. To test the hypothesis, we created Akita/miR-133aTg mice, a new strain of Akita where miR-133a is overexpressed in the heart, by crossbreeding male Akita with female cardiac-specific miR-133a transgenic mice. We validated Akita/miR-133aTg mice by genotyping and phenotyping (miR-133a levels in the heart). To determine whether miR-133a overexpression could prevent cardiac remodeling and cardiomyopathy, we evaluated cardiac fibrosis, hypertrophy, and dysfunction (P-V loop) in 13–15 week male WT, Akita, Akita/miR-133aTg, and miR-133aTg mice. Our results revealed that miR-133a overexpression in the Akita heart prevents DM-induced cardiac fibrosis (reduced collagen deposition), hypertrophy (decreased beta-myosin heavy chain), and impaired contractility (downregulated calcium handling protein sarco-endoplasmic reticulum-ATPase-2a). These results demonstrate that increased levels of miR-133a in the DM heart could prevent cardiac remodeling. Our P-V loop analysis showed a trend of decreased cardiac output, stroke volume, and ± dp/dt in Akita, which were blunted in Akita/miR-133aTg heart. These findings suggest that 13–15 week Akita heart undergoes adverse remodeling toward cardiomyopathy, which is prevented by miR-133a overexpression. In addition, increased cardiac miR-133a in the Akita heart did not change blood glucose levels but decreased lipid accumulation in the heart, suggesting inhibition of metabolic remodeling in the heart. Thus, miR-133a could be a promising therapeutic candidate to prevent DMCM.
Persistent, unresolved inflammation in adipose tissue is a major contributor to obesity-associated metabolic complications. However, the molecular links between lipid-overloaded adipocytes and inflammatory immune cells in obese adipose tissues remain elusive. Here we identified adipocyte-secreted microRNA-34a (miR-34a) as a key mediator through its paracrine actions on adipose-resident macrophages. The expression of miR-34a in adipose tissues was progressively increased with the development of dietary obesity. Adipose-selective or adipocyte-specific miR-34a–KO mice were resistant to obesity-induced glucose intolerance, insulin resistance, and systemic inflammation, and this was accompanied by a significant shift in polarization of adipose-resident macrophages from proinflammatory M1 to antiinflammatory M2 phenotype. Mechanistically, mature adipocyte-secreted exosomes transported miR-34a into macrophages, thereby suppressing M2 polarization by repressing the expression of Krüppel-like factor 4 (Klf4). The suppressive effects of miR-34a on M2 polarization and its stimulation of inflammatory responses were reversed by ectopic expression of Klf4 in both bone marrow–derived macrophages and adipose depots of obese mice. Furthermore, increased miR-34a expression in visceral fat of overweight/obese subjects correlated negatively with reduced Klf4 expression, but positively with the parameters of insulin resistance and metabolic inflammation. In summary, miR-34a was a key component of adipocyte-secreted exosomal vesicles that transmitted the signal of nutrient overload to the adipose-resident macrophages for exacerbation of obesity-induced systemic inflammation and metabolic dysregulation. © 2018 American Society for Clinical Investigation.All right reserved.
Exosomes, extracellular vesicles with diameters ranging from 30 to 150 nm, are widely present in various body fluids. Recently, microRNAs (miRNAs) have been identified in exosomes, the biogenesis, release, and uptake of which may involve the endosomal sorting complex required for transport (ESCRT complex) and relevant proteins. After release, exosomes are taken up by neighboring or distant cells, and the miRNAs contained within modulate such processes as interfering with tumor immunity and the microenvironment, possibly facilitating tumor growth, invasion, metastasis, angiogenesis and drug resistance. Therefore, exosomal miRNAs have a significant function in regulating cancer progression. Here, we briefly review recent findings regarding tumor-derived exosomes, including RNA sorting and delivering mechanism. We then describe the intercommunication occurring between different cells via exosomal miRNAs in tumor microenvironmnt, with impacts on tumor proliferation, vascularization, metastasis and other biological characteristics. Finally, we highlight the potential role of these molecules as biomarkers in cancer diagnosis and prognosis and tumor resistance to therapeutics.
Chronic inflammation accompanies obesity and limits subcutaneous white adipose tissue (WAT)
expandability, accelerating the development of insulin resistance and type 2 diabetes mellitus
(T2DM). MicroRNAs influence expression of many metabolic genes in fat cells, but
physiological roles in WAT remain poorly characterized. Here, we report expression of the
microRNA miR-30a in subcutaneous WAT corresponds with insulin sensitivity in obese mice
and humans. To examine the hypothesis that restoration of miR-30a expression in WAT
improves insulin sensitivity, we injected adenovirus (Adv) expressing miR-30a into the
subcutaneous fat pad of diabetic mice. Exogenous miR-30a expression in the subcutaneous WAT
depot of obese mice coupled improved insulin sensitivity and increased energy expenditure with
decreased ectopic fat deposition in the liver and reduced WAT inflammation. High throughput
proteomic profiling and RNA-Seq suggested miR-30a targets the transcription factor STAT1 to
limit the actions of the pro-inflammatory cytokine interferon-gamma (IFNγ), that would
otherwise restrict WAT expansion and decrease insulin sensitivity. We further demonstrated
miR-30a opposes the actions of IFNγ, suggesting an important role for miR-30a in defending
adipocytes against pro-inflammatory cytokines that reduce peripheral insulin sensitivity.
Together, our data identify a critical molecular signaling axis, elements of which are involved in
uncoupling obesity from metabolic dysfunction.
Both fibrosis and cirrhosis of the liver are the end results of most kinds of chronic liver damage and represent a common but difficult clinical challenge throughout the world. The inhibition of the fibrogenic, proliferative, and migratory effects of hepatic stellate cells (HSCs) has become an experimental therapy for preventing and even reversing hepatic fibrosis. Furthermore, a complete understanding of the function of non-coding RNA-mediated epigenetic mechanisms in HSC activation may improve our perception of liver fibrosis pathogenesis. This review focuses on the evolving view of the molecular mechanisms by which HSC activation by miR-29a signaling may moderate the profibrogenic phenotype of these cells, thus supporting the use of miR-29a agonists as a potential therapy for treating liver fibrosis in the future.
Background/objectives:
Obesity is a pandemic disorder that is characterized by accumulation of adipose tissue and chronic low-grade inflammation that is driven primarily by adipose tissue macrophages (ATMs). While ATM polarization from pro-(M1) to anti-(M2) inflammatory phenotype influences insulin sensitivity and energy expenditure, the mechanisms of such a switch are unclear. In the current study, we identified epigenetic pathways including microRNAs (miR) in ATMs that regulate obesity-induced inflammation.
Subjects/methods:
Male C57BL/6J mice were fed normal chow diet (NCD) or high-fat diet (HFD) for 16 weeks to develop lean and diet-induced obese mice, respectively. Transcriptome microarrays, microRNA microarrays, and MeDIP-Seq were performed on ATMs isolated from visceral fat. Pathway analysis and bone marrow-derived macrophage (BMDM) transfections further allowed computational and functional analysis of miRNA-mediated ATM polarization.
Results:
ATMs from HFD-fed mice were skewed toward M1 inflammatory phenotype. Concurrently, the expression of miRs 30a-5p, 30c-5p, and 30e-5p was downregulated in ATMs from HFD mice when compared to mice fed NCD. The miR-30 family was shown to target Delta-like-4, a Notch1 ligand, whose expression was increased in HFD ATMs. Inhibition of miR-30 in conditioned BMDM triggered Notch1 signaling, pro-inflammatory cytokine production, and M1 macrophage polarization. In addition, DNA hypermethylation was observed in mir30-associated CpG islands, suggesting that HFD downregulates miR-30 through epigenetic modifications.
Conclusions:
HFD-induced obesity downregulates miR-30 by DNA methylation thereby inducing Notch1 signaling in ATMs and their polarization to M1 macrophages. These findings identify miR-30 as a regulator of pro-inflammatory ATM polarization and suggest that miR-30 manipulation could be a therapeutic target for obesity-induced inflammation.
Introduction
Exosomes are closed-membrane nanovesicles that are secreted by a variety of cells and exist in most body fluids. Recent studies have demonstrated the potential of exosomes as natural vehicles that target delivery of functional small RNA and chemotherapeutics to diseased cells.
Methods
In this study, we introduce a new approach for the targeted delivery of exosomes loaded with functional miR-26a to scavenger receptor class B type 1-expressing liver cancer cells. The tumor cell-targeting function of these engineered exosomes was introduced by expressing in 293T cell hosts, the gene fusion between the transmembrane protein of CD63 and a sequence from Apo-A1. The exosomes harvested from these 293T cells were loaded with miR-26a via electroporation.
Results
The engineered exosomes were shown to bind selectively to HepG2 cells via the scavenger receptor class B type 1–Apo-A1 complex and then internalized by receptor-mediated endocytosis. The release of miR-26a in exosome-treated HepG2 cells upregulated miR-26a expression and decreased the rates of cell migration and proliferation. We also presented evidence that suggest cell growth was inhibited by miR-26a-mediated decreases in the amounts of key proteins that regulate the cell cycle.
Conclusion
Our gene delivery strategy can be adapted to treat a broad spectrum of cancers by expressing proteins on the surface of miRNA-loaded exosomes that recognize specific biomarkers on the tumor cell.
Background
The global pandemic of obesity has led to an increased risk for prediabetes and type-2 diabetes (T2D). The aims of the current project are: (1) to evaluate the effect of a 22-week family based intervention program, including supervised exercise, on insulin resistance syndrome (IRS) risk in children with a high risk of developing T2D and (2) to identify the profile of microRNA in circulating exosomes and in peripheral blood mononuclear cells in children with a high risk of developing T2D and its response to a multidisciplinary intervention program including exercise. MethodsA total of 84 children, aged 8–12 years, with a high risk of T2D will be included and randomly assigned to control (N = 42) or intervention (N = 42) groups. The control group will receive a family based lifestyle education and psycho-educational program (2 days/month), while the intervention group will attend the same lifestyle education and psycho-educational program plus the exercise program (3 days/week, 90 min per session including warm-up, moderate to vigorous aerobic activities, and strength exercises). The following measurements will be evaluated at baseline prior to randomization and after the intervention: fasting insulin, glucose and hemoglobin A1c; body composition (dual-energy X-ray absorptiometry); ectopic fat (magnetic resonance imaging); microRNA expression in circulating exosomes and in peripheral blood mononuclear cells (MiSeq; Illumina); cardiorespiratory fitness (cardiopulmonary exercise testing); dietary habits and physical activity (accelerometry). DiscussionPrevention and identification of children with a high risk of developing T2D could help to improve their cardiovascular health and to reduce the comorbidities associated with obesity. Trial registrationClinicalTrials.gov, ID: NCT03027726. Registered on 16 January 2017.
A number of microRNAs (miRNAs, miRs) have been shown to play a role in skeletal muscle atrophy, but their role is not completely understood. Here we show that miR-29b promotes skeletal muscle atrophy in response to different atrophic stimuli in cells and in mouse models. miR-29b promotes atrophy of myotubes differentiated from C2C12 or primary myoblasts, and conversely, its inhibition attenuates atrophy induced by dexamethasone (Dex), TNF-alpha and H2O2 treatment. Targeting of IGF-1 and PI3K(p85alpha) by miR-29b is required for induction of muscle atrophy. In vivo, miR-29b overexpression is sufficient to promote muscle atrophy while inhibition of miR-29b attenuates atrophy induced by denervation and immobilization. These data suggest that miR-29b contributes to multiple types of muscle atrophy via targeting of IGF-1 and PI3K(p85alpha), and that suppression of miR-29b may represent a therapeutic approach for muscle atrophy induced by different stimuli.
The intrinsic ability of neurogenesis after stroke has been proven weak, which results in insufficient repair of injury in the nerve system. Recent studies suggest multiple microRNAs (miRNAs) are involved in the neuroremodeling process. Targeted miRNAs delivery for amplification of neurogenesis is promising in promoting the prognosis after ischemia. Here we showed that modified exosomes, with rabies virus glycoprotein (RVG) fused to exosomal protein lysosome-associated membrane glycoprotein 2b (Lamp2b), could efficiently deliver miR-124 to the infarct site. Systemic administration of RVG-exosomes loaded with miR-124 promoted cortical neural progenitors to obtain neuronal identity and protect against ischemic injury by robust cortical neurogenesis. Our study suggests that RVG-exosomes can be utilized therapeutically for the targeted delivery of gene drugs to the brain, thus having great potential for clinical applications.
Nonalcoholic steatohepatitis (NASH) is a liver disease associated with significant morbidity. Kupffer cells (KCs) produce endogenous miR-690 and, via exosome secretion, shuttle this miRNA to other liver cells, such as hepatocytes, recruited hepatic macrophages (RHMs), and hepatic stellate cells (HSCs). miR-690 directly inhibits fibrogenesis in HSCs, inflammation in RHMs, and de novo lipogenesis in hepatocytes. When an miR-690 mimic is administered to NASH mice in vivo, all the features of the NASH phenotype are robustly inhibited. During the development of NASH, KCs become miR-690 deficient, and miR-690 levels are markedly lower in mouse and human NASH livers than in controls. KC-specific KO of miR-690 promotes NASH pathogenesis. A primary target of miR-690 is NADK mRNA, and NADK levels are inversely proportional to the cellular miR-690 content. These studies show that KCs play a central role in the etiology of NASH and raise the possibility that miR-690 could emerge as a therapeutic for this condition.
Scope:
Phenotypic switch of macrophage polarization in adipose tissue has been associated with obesity-induced adipose tissue inflammation (OATI). Therefore, we aimed to explore the possible mechanism of adipocytes-derived exosomes (ADEs) carrying microRNA-1224 (miR-1224) in M2 macrophage polarization of OATI.
Methods and results:
We developed miR-1224-knockout (miR-1224-KO) mice for this study, and isolated primary adipocytes from high-fat diet (HFD) or normal diet (SD)-fed mice. ADEs were extracted and cocultured with bone marrow-derived macrophages (BMDMs). The macrophagic crown-like structures (CLS) and M1 and M2 phenotype macrophages in epididymal white adipose tissue (epiWAT) were observed by immunohistochemistry and flow cytometry. The obtained data indicated that miR-1224 was highly expressed in adipose tissues and adipocytes of obese mice. miR-1224 knockout decreased CLS number and increased M2 macrophages polarization in epiWAT. In addition, miR-1224 could be transferred to BMDMs via ADEs, which targeted musashi RNA binding protein 2 (MSI2) expression and inactivated Wnt/β-catenin pathway, inhibiting macrophage M2 polarization and promoting inflammatory factor release.
Conclusion:
Exosomal miR-1224 derived by adipocytes targets MSI2 and blocks the Wnt/β-catenin pathway, which inhibits macrophage M2 polarization and promotes inflammatory factor release, ultimately promoting OATI. This article is protected by copyright. All rights reserved.
Exosomes are nanoparticles secreted by all cell types and are a large component of the broader class of nanoparticles termed extracellular vesicles (EVs). Once secreted, exosomes gain access to the interstitial space and ultimately the circulation, where they exert local paracrine or distal systemic effects. Because of this, exosomes are important components of an intercellular and intraorgan communication system capable of carrying biologic signals from one cell type or tissue to another. The exosomal cargo consists of proteins, lipids, miRNAs, and other RNA species, and many of the biologic effects of exosomes have been attributed to miRNAs. Exosomal miRNAs have also been used as disease biomarkers. The field of exosome biology and metabolism is rapidly expanding, with new discoveries and reports appearing on a regular basis, and it is possible that potential therapeutic approaches for the use of exosomes or miRNAs in metabolic diseases will be initiated in the near future.
Insulin resistance is a major pathophysiologic defect in type 2 diabetes and obesity, while anti-inflammatory M2-like macrophages are important in maintaining normal metabolic homeostasis. Here, we show that M2 polarized bone marrow-derived macrophages (BMDMs) secrete miRNA-containing exosomes (Exos), which improve glucose tolerance and insulin sensitivity when given to obese mice. Depletion of their miRNA cargo blocks the ability of M2 BMDM Exos to enhance insulin sensitivity. We found that miR-690 is highly expressed in M2 BMDM Exos and functions as an insulin sensitizer both in vivo and in vitro. Expressing an miR-690 mimic in miRNA-depleted BMDMs generates Exos that recapitulate the effects of M2 BMDM Exos on metabolic phenotypes. Nadk is a bona fide target mRNA of miR-690, and Nadk plays a role in modulating macrophage inflammation and insulin signaling. Taken together, these data suggest miR-690 could be a new therapeutic insulin-sensitizing agent for metabolic disease.
Significance
The beneficial metabolic effects of exercise are mediated at least in part by the release of soluble factors by the muscles. Exosomes, small vesicles that facilitate the exchange of biological components among cells and tissues, may constitute one of these factors. Here, we show that exercise triggers the release of exosomes by the trained muscle, carrying a specific miRNA signature that induces gene expression changes in the liver, finally contributing to increased insulin sensitivity. Molecular characterization of exercise-induced exosomal miRNAs and their effects may drive the design of novel therapeutic strategies to alleviate insulin resistance and other aging-related ailments in an increasingly older society.
Exosomes are secreted nanovesicles that are able to transfer their cargo (such as miRNAs) between cells. To determine to what extent exosomes and exosomal miRNAs are involved in the pathogenesis, progression and diagnosis of viral infections. The scientific literature (PubMed and Google Scholar) was searched from 1970 to 2019. The complex biogenesis of exosomes and miRNAs was reviewed. Exosomes contain both viral and host miRNAs that can be used as diagnostic biomarkers for viral diseases. Viral proteins can alter miRNAs, and conversely miRNAs can alter the host response to viral infections in a positive or negative manner. It is expected that exosomal miRNAs will be increasingly used for diagnosis, monitoring and even treatment of viral infections.
Engineered exosomes have become popular drug delivery carriers for cancer treatment. This is partially due to the interesting property, i.e. exosome organotropism, which plays important role on organ distribution post systemic administration. Here, we demonstrated that breast cancer (MDA-MB-231) cell-derived exosomes (231-Exo) could be specifically internalized by non-small cell lung cancer cells via specific interaction between overexpressed integrin β4 (on exosomes) and surfactant protein C (SPC) on the cancer cells. We showed that 231-Exo was capable of recognizing A549 cells in blood and effectively escaping from immune surveillance system in vitro. Once loaded with microRNA molecule in the exosome carriers, the resulting, miRNA-126 loaded 231-Exo (miRNA-231-Exo) strongly suppressed A549 lung cancer cell proliferation and migration through the interruption of PTEN/PI3K/AKT signaling pathway. Intravenous administration of the miRNA-126 laden exosomes led to an effective lung homing effect in mice. When tested in a lung metastasis model, miRNA-231-Exo resulted in efficacious effect in inhibiting the formulation of lung metastasis in mice. Collectively, our data demonstrated the possibility for using organotropism feature of exosomes in nanocarrier design, generating potent anti-metastasis effect in a mouse model.
Background:
Fracture healing is a complex process, and patients with fracture will undergo non-union or compromised regeneration. MicroRNA (miR)-342-5p is a Notch downstream molecule, and its roles in fracture healing remain unclear. We aimed to explore the functional roles of miR-342-5p in osteoblasts as well as the underlying mechanisms.
Methods:
The expression of miR-342-5p in differentiation of MC3T3-E1 cells or hMSCs was examined by quantitative reverse transcription PCR (qRT-PCR). The effects of aberrantly expressed miR-342-5p on cell proliferation, apoptosis, migration, and expressions of proteins associated with proliferation and osteogenic differentiation were determined by Cell Counting Kit-8, trypan blue staining, flow cytometry, Transwell assay, Western blot and qRT-PCR assays, respectively. The downstream factor and the target genes of miR-342-5p as well as the involvements of the mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway were finally assessed.
Results:
miR-342-5p level was decreased during differentiation of MC3T3-E1 cells or hMSCs. After cell transfection, miR-342-5p overexpression significantly reduced cell viability, induced apoptosis, inhibited proliferation, migration and differentiation, and down-regulated Bmp7 expression. Subsequent experiments showed the effects of miR-342-5p inhibition on MC3T3-E1 cells were abrogated by Bmp7 knockdown. Additionally, COL4A6 and Bmp2 were predicated as target genes of miR-342-5p. Finally, phosphorylated levels of MEK and ERK were increased by miR-342-5p inhibition via up-regulating Bmp7 expression.
Conclusion:
miR-342-5p inhibition promoted proliferation, migration and differentiation of osteoblasts via regulating Bmp7, along with activation of the MEK/ERK pathway.
Objective:
This study aimed to investigate cellular sources of microRNAs (miRNA) within adipose tissue and the impact of obesity on miRNA expression, as well as to examine targets of miRNAs.
Methods:
miRNA expression by quantitative polymerase chain reaction was examined in adipocytes, adipose tissue macrophages (ATM), and peripheral blood mononuclear cells from and individuals with normal weight and with obesity. Differentiated 3T3-L1 adipocytes were cocultured with macrophages, and 3T3-L1 and differentiated human mesenchymal stem cells were transfected with miR-155, with peroxisome proliferator-activated receptor gamma (PPAR-γ) and solute carrier family 2 member 4 (GLUT4) abundance measured via Western blot analysis.
Results:
Abundance of miR-155 and miR-210 was increased in ATM of participants with obesity by 6.7-fold and 2.9-fold (P = 0.002 and P = 0.013, respectively). miR-130b expression was increased 1.8-fold in ATM and 4.3-fold in adipocytes from participants with obesity (P = 0.007 and P = 0.02, respectively). PPARG mRNA expression decreased 32% (P = 0.044) in adipocytes from individuals with obesity. In 3T3-L1 cells exposed to macrophages, PPARG expression decreased 99.4% (P = 0.02). PPAR-γ protein content declined 75% (P = 0.001) in 3T3-L1 cells transfected with miR-155. GLUT4 protein levels were reduced by 55% (P = 0.021) in differentiated human mesenchymal stem cells exposed to miR-155.
Conclusions:
Adipose tissue miRNAs are influenced in a cell type-specific fashion by obesity, with macrophage miR-155 potentially impacting neighboring adipocytes.
Immune cells residing in white adipose tissue have been highlighted as important factors contributing to the pathogenesis of metabolic diseases, but the molecular regulators that drive adipose tissue immune cell remodeling during obesity remain largely unknown. Using index and transcriptional single-cell sorting, we comprehensively map all adipose tissue immune populations in both mice and humans during obesity. We describe a novel and conserved Trem2+ lipid-associated macrophage (LAM) subset and identify markers, spatial localization, origin, and functional pathways associated with these cells. Genetic ablation of Trem2 in mice globally inhibits the downstream molecular LAM program, leading to adipocyte hypertrophy as well as systemic hypercholesterolemia, body fat accumulation, and glucose intolerance. These findings identify Trem2 signaling as a major pathway by which macrophages respond to loss of tissue-level lipid homeostasis, highlighting Trem2 as a key sensor of metabolic pathologies across multiple tissues and a potential therapeutic target in metabolic diseases.
Obesity-associated insulin resistance is a forerunner of type 2 diabetes. Macrophages reside within adipose tissue (ATMs) have been reported to regulate insulin sensitivity through secreting miRNAs containing exosomes. Here, we show that miR-29a is increased in obese ATMs derived exosomes (ATMs-Exos) and can be transferred into adipocytes, myocytes and hepatocytes causing insulin resistance in vitro and in vivo. Administration of obese ATMs-Exos impairs insulin sensitivity of lean mice. While knockdown miR-29a level in obese ATM-Exos blunts this effect. PPAR-δ is identified to function as downstream target of miR-29a in regulating insulin resistance. PPAR-δ agonist GW501516 partially rescued the insulin resistance induced by miR-29a. Taken together, these findings suggest that ATMs derived exosomal miR-29a could regulate obesity-associated insulin resistance, which may serve as a potential therapeutic target for obesity-associated type 2 diabetes.
Type 1 diabetes, has been recognized as an autoimmune disease. Like other immunological conditions, regulation of immune response is a key strategy to control the autoimmunity in diabetic patients. Mesenchymal stem cells have been shown to have a distinct potential in modulating the immune reactions. However, treatment with stem cells is combined with concerns about safety issues. To overcome these concerns, in this study, we have utilized the regenerative potential of exosomes isolated from menstrual blood‐derived mesenchymal stem cells to restore the β‐cell mass and insulin production in type 1 diabetes. Exosomes are nanovesicles containing various cargos involved in cellular communications. Streptozotocin was used to induce islet destruction and diabetes in male Wistar rats. Then, exosomes were intravenously injected into animals at different time points and in a single or repeated therapeutic doses. After about 6 weeks, animals were euthanized and the pancreas was analyzed for the presence of the regenerated β islets as well as the insulin secretion. The non‐fasting blood glucose and the serum insulin level were also monitored during the study. Our results represented that menstrual blood‐derived mesenchymal stem cell‐derived exosomes enhance the β‐cell mass and insulin production in the pancreas of diabetic animals that received repeated doses of exosomes. Immunohistochemistry analysis also confirmed the presence of insulin in the islets of treated animals. Further investigations proposed that exosomes induce the islet regeneration through pancreatic and duodenal homeobox 1 pathway. The exosome tracking also revealed the homing of injected exosomes to the pancreas. The figure highlights the strategy and results of the study.
Rationale:
Exercise training, in addition to reducing cardiovascular risk factors, confers direct protection against myocardial ischemia/reperfusion injury and has been associated with improved heart attack survival in humans. However, the underlying mechanisms of exercise-afforded cardioprotection are still unclear.
Objective:
To investigate the role of exercise-derived circulating exosomes in cardioprotection and the molecular mechanisms involved.
Methods and results:
Circulating exosomes were isolated from the plasma of volunteers with or without exercise training and rats subjected to 4-week swim exercise or sedentary littermates 24 hours after the last training session. Although the total circulating exosome level did not change significantly in exercised subjects 24 hours post-exercise compared with the sedentary control, the isolated plasma exosomes from exercised rats afforded remarkable protection against myocardial ischemia/reperfusion injury. miRNA sequencing combined with quantitative reverse transcription polymerase chain reaction validation identified 12 differentially expressed miRNAs from the circulating exosomes of exercised rats, among which miR-342-5p stood out as the most potent cardioprotective molecule. Importantly, the cardioprotective effects and the elevation of exosomal miR-342-5p were also observed in exercise-trained human volunteers. Moreover, inhibition of miR-342-5p significantly blunted the protective effects of exercise-derived circulating exosomes in hypoxia/reoxygenation cardiomyocytes; in vivo cardiac-specific inhibition of miR-342-5p through serotype 9 adeno-associated virus-mediated gene delivery attenuated exercise-afforded cardioprotection in myocardial ischemia/reperfusion rats. Mechanistically, miR-342-5p inhibited hypoxia/reoxygenation-induced cardiomyocyte apoptosis via targeting Caspase 9 and Jnk2; it also enhanced survival signaling (p-Akt) via targeting phosphatase gene Ppm1f. Of note, exercise training or laminar shear stress directly enhanced the synthesis of miR-342-5p in endothelial cells.
Conclusions:
Our findings reveal a novel endogenous cardioprotective mechanism that long-term exercise-derived circulating exosomes protect the heart against myocardial ischemia/reperfusion injury via exosomal miR-342-5p.
Insulin resistance is the major pathological characteristic of type 2 diabetes and the elderly often develop insulin resistance. However, the deep-seated mechanisms for aging-related insulin resistance remain unclear. Here, we showed that nano-sized exosomes released by bone marrow mesenchymal stem cells (BM-MSCs) of aged mice could be taken up by adipocytes, myocytes, and hepatocytes, resulting in insulin resistance both in vivo and in vitro. Using microRNA (miRNA) array assays, we found that the amount of miR-29b-3p was dramatically increased in exosomes released by BM-MSCs of aged mice. Mechanistically, SIRT1 (sirtuin 1) was identified to function as the downstream target of exosomal miR-29b-3p in regulating insulin resistance. Notably, utilizing an aptamer-mediated nanocomplex delivery system, down-regulated the level of miR-29b-3p in BM-MSCs-derived exosomes significantly ameliorated the insulin resistance of aged mice. Meanwhile, BM-MSCs-specific overexpression of miR-29b-3p induced insulin resistance in young mice. Taken together, these findings suggested that BM-MSCs-derived exosomal miR-29b-3p could modulate aging-related insulin resistance, which may serve as a potential therapeutic target for aging-associated insulin resistance.
Type 1 diabetes is an autoimmune disease initiated by the invasion of pancreatic islets by immune cells that selectively kill the beta cells. We found that rodent and human T lymphocytes release exosomes containing the microRNAs (miRNAs) miR-142-3p, miR-142-5p, and miR-155, which can be transferred in active form to beta cells favoring apoptosis. Inactivation of these miRNAs in recipient beta cells prevents exosome-mediated apoptosis and protects non-obese diabetic (NOD) mice from diabetes development. Islets from protected NOD mice display higher insulin levels, lower insulitis scores, and reduced inflammation. Looking at the mechanisms underlying exosome action, we found that T lymphocyte exosomes trigger apoptosis and the expression of genes involved in chemokine signaling, including Ccl2, Ccl7, and Cxcl10, exclusively in beta cells. The induction of these genes may promote the recruitment of immune cells and exacerbate beta cell death during the autoimmune attack. Our data point to exosomal-miRNA transfer as a communication mode between immune and insulin-secreting cells.
The expansion of adipose tissue in obesity is accompanied by the accumulation of immune cells that contribute to a state of low‐grade, chronic inflammation and dysregulated metabolism. Adipose tissue macrophages (ATMs) represent the most abundant class of leukocytes in adipose tissue (AT) and are involved in the regulation of several regulatory physiological processes such as tissue remodeling and insulin sensitivity. With progressive obesity, ATMs are key mediators of meta‐inflammation, insulin resistance, and impairment of adipocyte function. While macrophage recruitment from blood monocytes is a critical component of the generation of adipose tissue inflammation, new studies have revealed a role for ATM proliferation in the early stages of obesity and in sustaining adipose tissue inflammation. In addition, studies have revealed a more complex range of macrophage activation states than the previous M1/M2 model and the existence of different macrophage profiles between human and animal models. This review will summarize the current understanding of the regulatory mechanisms of ATM function in relation to obesity, to type 2 diabetes, to depot of origin, and to other leukocytes such as adipose tissue dendritic cells with hopes of emphasizing the regulatory nodes that can potentially be targeted to prevent and treat obesity‐related metabolic disorders.
This article is protected by copyright. All rights reserved.
Transforming growth factor-β1 (TGF-β1)-induced epithelial to mesenchymal transition (EMT) and renal fibrosis plays critical role in the development and progression of diabetic nephropathy (DN). Our study aimed to determine the detailed roles of miR-133b &miR-199b on TGF-β1-induced EMT &renal fibrosis in DN and its underlying mechanism. The expressions of miR-133b &miR-199b in OLETF rats, LETO rats&TGF-β1-treated human proximal tubule cell line (HK-2) were examined by qRT-PCR. Inhibition of miR-133b or miR-199b was realized in cells by transfection of lentivirus containing miR-133b inhibit or miR-199b inhibitor. The expression levels of collagen I (COL I, fibronectin (FN), α-smooth muscle actin (α-SMA), E-cadherin & sirtuin 1 (SIRT1) were detected by western blot and immunohistochemistry. Masson staining was conducted to estimate the degree of renal fibrosis. The interaction between SIRT1 and miR-133b, miR-199b was explored by luciferase reporter assay and RNA immunoprecipitation (RIP) assay.miR-133b and miR-199b were highly expressed in the renal cortex of diabetic OLETF rats and TGF-β1-treated HK-2 cells. EMT and renal fibrosis were induced in diabetic OLETF rats and TGF-β1-treated HK-2 cells. Inhibition of miR-133b and miR-199b attenuated EMT and renal fibrosis in diabetic OLETF rats and TGF-β1-treated HK-2 cells. In addition, SIRT1 was identified as a target of miR-133b &miR-199b in HK-2 cells. SIRT1 knockdown dramatically reversed the suppression on TGF-β1-induced EMT and renal fibrosis in HK-2 cells mediated by anti-miR-133b or anti-miR-199.Inhibition of miR-133b &miR-199b attenuated TGF-β1-induced EMT &renal fibrosis by upregulating SIRT1 shows that using different miRNAs is a potential strategy for the future treatment of DN.
In human adipose tissue and obesity, miR-99a expression is negatively correlated with inflammation. Therefore, the present study investigated the role of miR-99a in macrophage phenotype activation and adipose tissue inflammation. M2 BMDMs showed a significant increase in miR-99a expression when compared to the M0 and M1 phenotypes. Phenotype-switching experiments established an association between upregulated miR-99a expression and the M2 phenotype. Overexpression of miR-99a prevented M1 phenotype activation and attenuated bactericidal activity. Likewise, knockdown of miR-99a abolished M2 phenotype activation. By means of in silico target prediction tools and a luciferase reporter assay, TNFα was identified as a direct target of miR-99a. Knockdown of TNFα recapitulated the effect of miR-99a overexpression in M1 BMDMs. In a db/db mice model, miR-99a expression was reduced in eWAT and F4/80+ ATMs. Systemic overexpression of miR-99a in db/db mice attenuated adipocyte hypertrophy with increased CD301 and reduced CD86 immunostaining. Flow cytometry analysis also showed an increased M2 and a reduced M1 macrophage population. Mimics of miR-99a also improved the diabetic dyslipidemia and insulin signaling in eWAT and liver, with an attenuated expression of gluconeogenesis and cholesterol metabolism genes in the liver. Furthermore, adoptive transfer of miR-99a-overexpressing macrophages in the db/db mice recapitulated in vivo miR-99a mimic effects with increased M2 and reduced M1 macrophage populations and improved systemic glucose, insulin sensitivity, and insulin signaling in the eWAT and liver. The present study demonstrates that miR-99a mimics can regulate macrophage M1 phenotype activation by targeting TNFα. miR-99a therapeutics in diabetic mice reduces the adipose tissue inflammation and improves insulin sensitivity.
Significance
Obesity has reached pandemic levels, prompting the need for novel therapeutics. The immune system has been suggested to be critically linked to metabolic health, leading to the prospect of immune-directed therapies. We report that obese fat tissue contains multiple distinct populations of macrophages with unique tissue distributions, transcriptomes, chromatin landscapes, and functions. These results provide a higher resolution of the cellular and functional heterogeneity within adipose macrophages and provide a framework within which to develop new immune-directed therapies for the treatment of obesity and related inflammatory comorbidities.
Extracellular vesicles are a heterogeneous group of cell-derived membranous structures comprising exosomes and microvesicles, which originate from the endosomal system or which are shed from the plasma membrane, respectively. They are present in biological fluids and are involved in multiple physiological and pathological processes. Extracellular vesicles are now considered as an additional mechanism for intercellular communication, allowing cells to exchange proteins, lipids and genetic material. Knowledge of the cellular processes that govern extracellular vesicle biology is essential to shed light on the physiological and pathological functions of these vesicles as well as on clinical applications involving their use and/or analysis. However, in this expanding field, much remains unknown regarding the origin, biogenesis, secretion, targeting and fate of these vesicles.
MiRNAs are regulatory molecules that can be packaged into exosomes and secreted from cells. Here, we show that adipose tissue macrophages (ATMs) in obese mice secrete miRNA-containing exosomes (Exos), which cause glucose intolerance and insulin resistance when administered to lean mice. Conversely, ATM Exos obtained from lean mice improve glucose tolerance and insulin sensitivity when administered to obese recipients. miR-155 is one of the miRNAs overexpressed in obese ATM Exos, and earlier studies have shown that PPARγ is a miR-155 target. Our results show that miR-155KO animals are insulin sensitive and glucose tolerant compared to controls. Furthermore, transplantation of WT bone marrow into miR-155KO mice mitigated this phenotype. Taken together, these studies show that ATMs secrete exosomes containing miRNA cargo. These miRNAs can be transferred to insulin target cell types through mechanisms of paracrine or endocrine regulation with robust effects on cellular insulin action, in vivo insulin sensitivity, and overall glucose homeostasis.
Obesity is characterized by the chronic low-grade activation of the innate immune system. In this respect, macrophage-elicited metabolic inflammation and adipocyte-macrophage interaction has a primary importance in obesity. Large amounts of macrophages are accumulated by different mechanisms in obese adipose tissue. Hypertrophic adipocyte-derived chemotactic monocyte chemoattractant protein-1 (MCP-1)/C-C chemokine receptor 2 (CCR2) pathway also promotes more macrophage accumulation into the obese adipose tissue. However, increased local extracellular lipid concentrations is a final mechanism for adipose tissue macrophage accumulation. A paracrine loop involving free fatty acids and tumor necrosis factor-alpha (TNF-alpha) between adipocytes and macrophages establishes a vicious cycle that aggravates inflammatory changes in the adipose tissue. Adipocyte-specific caspase-1 and production of interleukin-1beta (IL-1beta) by macrophages; both adipocyte and macrophage induction by toll like receptor-4 (TLR4) through nuclear factor-kappaB (NF-kappaB) activation; free fatty acid-induced and TLR-mediated activation of c-Jun N-terminal kinase (JNK)-related pro-inflammatory pathways in CD11c+ immune cells; are effective in macrophage accumulation and in the development of adipose tissue inflammation. Old adipocytes are removed by macrophages through trogocytosis or sending an “eat me” signal. The obesity-induced changes in adipose tissue macrophage numbers are mainly due to increases in the triple-positive CD11b+ F4/80+ CD11c+ adipose tissue macrophage subpopulation. The ratio of M1-to-M2 macrophages is increased in obesity. Furthermore, hypoxia along with higher concentrations of free fatty acids exacerbates macrophage-mediated inflammation in obesity. The metabolic status of adipocytes is a major determinant of macrophage inflammatory output. Macrophage/adipocyte fatty-acid-binding proteins act at the interface of metabolic and inflammatory pathways. Both macrophages and adipocytes are the sites for active lipid metabolism and signaling.