Exosomes, microvesicles, and other extracellular vesicles-a Keystone Symposia report.

Extracellular vesicles (EVs) are small, lipid-bilayer-bound particles released by cells that can contain important bioactive molecules, including lipids, RNAs, and proteins. Once released in the extracellular environment, EVs can act as messengers locally as well as to distant tissues to coordinate tissue homeostasis and systemic responses. There is a growing interest in not only understanding the physiology of EVs as signaling particles but also leveraging them as minimally invasive diagnostic and prognostic biomarkers (e.g., they can be found in biofluids) and drug-delivery vehicles. On October 30-November 2, 2022, researchers in the EV field convened for the Keystone symposium "Exosomes, Microvesicles, and Other Extracellular Vesicles" to discuss developing standardized language and methodology, new data on the basic biology of EVs and potential clinical utility, as well as novel technologies to isolate and characterize EVs.

Pulmonary hypertension and insulin resistance: a mechanistic overview.

Pulmonary arterial hypertension (PAH) is a vascular remodeling disease, characterized by increased blood pressure levels in pulmonary circulation, leading to a restriction in the circulation flow and heart failure. Although the emergence of new PAH therapies has increased survival rates, this disease still has a high mortality and patients that receive diagnosis die within a few years. The pathogenesis of PAH involves multiple pathways, with a complex interaction of local and distant cytokines, hormones, growth factors, and transcription factors, leading to an inflammation that changes the vascular anatomy in PAH patients. These abnormalities involve more than just the lungs, but also other organs, and between these affected organs there are different metabolic dysfunctions implied. Recently, several publications demonstrated in PAH patients a disturbance in glucose metabolism, demonstrated by higher levels of glucose, insulin, and lipids in those patients. It is possible that a common molecular mechanism can have a significant role in this connection. In this regard, this narrative review intends to focus on the recent papers that mainly discuss the molecular determinants between insulin resistance (IR) associated PAH, which included obesity subclinical inflammation induced IR, PPAR gamma and Adiponectin, BMPR2, mitochondrial dysfunction and endoplasmic reticulum stress. Therefore, the following review will summarize some of the existing data for IR associated PAH, focusing on the better understanding of PAH molecular mechanisms, for the development of new translational therapies.

MicroRNA sequence codes for small extracellular vesicle release and cellular retention.

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 expression1-3. 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.

Helminth infection in mice improves insulin sensitivity via modulation of gut microbiota and fatty acid metabolism.

Intestinal helminths are prevalent in individuals who live in rural areas of developing countries, where obesity, type 2 diabetes, and metabolic syndrome are rare. In the present study, we analyzed the modulation of the gut microbiota in mice infected with the helminth Strongyloides venezuelensis, and fed either a standard rodent chow diet or high-fat diet (HFD). To investigate the effects of the microbiota modulation on the metabolism, we analyzed the expression of tight-junction proteins present in the gut epithelium, inflammatory markers in the serum and tissue and quantified glucose tolerance and insulin sensitivity and resistance. Additionally, the levels of lipids related to inflammation were evaluated in the feces and serum. Our results show that infection with Strongyloides venezuelensis results in a modification of the gut microbiota, most notably by increasing Lactobacillus spp. These modifications in the microbiota alter the host metabolism by increasing the levels of anti-inflammatory cytokines, switching macrophages from a M1 to M2 pattern in the adipose tissue, increasing the expression of tight junction proteins in the intestinal cells (thereby reducing the permeability) and decreasing LPS in the serum. Taken together, these changes correlate with improved insulin signaling and sensitivity, which could also be achieved with HFD mice treated with probiotics. Additionally, helminth infected mice produce higher levels of oleic acid, which participates in anti-inflammatory pathways. These results suggest that modulation of the microbiota by helminth infection or probiotic treatment causes a reduction in subclinical inflammation, which has a positive effect on the glucose metabolism of the host.

Blocking iNOS and endoplasmic reticulum stress synergistically improves insulin resistance in mice.

OBJECTIVE: Recent data show that iNOS has an essential role in ER stress in obesity. However, whether iNOS is sufficient to account for obesity-induced ER stress and Unfolded Protein Response (UPR) has not yet been investigated. In the present study, we used iNOS knockout mice to investigate whether high-fat diet (HFD) can still induce residual ER stress-associated insulin resistance. METHODS: For this purpose, we used the intraperitoneal glucose tolerance test (GTT), euglycemic-hyperinsulinemic clamp, western blotting and qPCR in liver, muscle, and adipose tissue of iNOS KO and control mice on HFD. RESULTS: The results of the present study demonstrated that, in HFD fed mice, iNOS-induced alteration in insulin signaling is an essential mechanism of insulin resistance in muscle, suggesting that iNOS may represent an important target that could be blocked in order to improve insulin sensitivity in this tissue. However, in liver and adipose tissue, the insulin resistance induced by HFD was only partially dependent on iNOS, and, even in the presence of genetic or pharmacological blockade of iNOS, a clear ER stress associated with altered insulin signaling remained evident in these tissues. When this ER stress was blocked pharmacologically, insulin signaling was improved, and a complete recovery of glucose tolerance was achieved. CONCLUSIONS: Taken together, these results reinforce the tissue-specific regulation of insulin signaling in obesity, with iNOS being sufficient to account for insulin resistance in muscle, but in liver and adipose tissue ER stress and insulin resistance can be induced by both iNOS-dependent and iNOS-independent mechanisms.

IKKε is key to induction of insulin resistance in the hypothalamus, and its inhibition reverses obesity.

IKK epsilon (IKKε) is induced by the activation of nuclear factor-κB (NF-κB). Whole-body IKKε knockout mice on a high-fat diet (HFD) were protected from insulin resistance and showed altered energy balance. We demonstrate that IKKε is expressed in neurons and is upregulated in the hypothalamus of obese mice, contributing to insulin and leptin resistance. Blocking IKKε in the hypothalamus of obese mice with CAYMAN10576 or small interfering RNA decreased NF-κB activation in this tissue, relieving the inflammatory environment. Inhibition of IKKε activity, but not TBK1, reduced IRS-1(Ser307) phosphorylation and insulin and leptin resistance by an improvement of the IR/IRS-1/Akt and JAK2/STAT3 pathways in the hypothalamus. These improvements were independent of body weight and food intake. Increased insulin and leptin action/signaling in the hypothalamus may contribute to a decrease in adiposity and hypophagia and an enhancement of energy expenditure accompanied by lower NPY and increased POMC mRNA levels. Improvement of hypothalamic insulin action decreases fasting glycemia, glycemia after pyruvate injection, and PEPCK protein expression in the liver of HFD-fed and db/db mice, suggesting a reduction in hepatic glucose production. We suggest that IKKε may be a key inflammatory mediator in the hypothalamus of obese mice, and its hypothalamic inhibition improves energy and glucose metabolism.