Javeed, Naureen

Abstract Chronically elevated circulating excess free fatty acids (ie, lipotoxicity) is a pathological process implicated in several metabolic disorders, including obesity-driven type 2 diabetes (T2D). Lipotoxicity exerts detrimental effects on pancreatic islet β-cells by reducing glucose-stimulated insulin secretion (GSIS), altering β-cell transcriptional identity, and promoting apoptosis. While β-cell-derived small extracellular vesicles (sEV) have been shown to contribute to β-cell failure in T2D, their specific role in lipotoxicity-mediated β-cell failure remains to be elucidated. In this work, we demonstrate that lipotoxicity enhances the release of sEVs from β-cells, which exhibit altered proteomic and lipidomic profiles. These palmitate (PAL)-exposed extracellular vesicles (EVs) induce β-cell dysfunction in healthy mouse and human islets and trigger significant islet transcriptional changes, including the upregulation of genes associated with the TGFβ/Smad3 pathway, as noted by RNA sequencing. Importantly, pharmacological inhibition of the TGFβI/II receptor improved PAL EV-induced β-cell dysfunction, underscoring their involvement in activating the TGFβ/Smad3 pathway during this process. We have comprehensively characterized lipotoxic β-cell sEVs and implicated their role in inducing β-cell functional failure in T2D. These findings highlight potential avenues for therapeutic interventions targeting sEV-mediated pathways to preserve β-cell health in metabolic disorders.

Abstract Extracellular vesicles (EVs) are key mediators of cell–cell communication and are involved in transferring specific biomolecular cargo to recipient cells to regulate their physiological functions. A major challenge in the understanding of EV function in vivo is the difficulty ascertaining the origin of the EV particles. The recent development of the “Snorkel-tag,” which includes EV-membrane-targeted CD81 fused to a series of extra-vesicular protein tags, can be used to mark EVs originating from a specific source for subsequent isolation and characterization. We developed an in vivo mouse model, termed “CAGS-Snorkel,” which expresses the Snorkel-tag under the control of the Cre-lox system, and crossed this mouse with either Prx1-Cre (mesenchymal progenitors) or Ocn-Cre (osteoblasts/osteocytes) and isolated Snorkel-tagged EVs from the mouse bone marrow plasma using a magnetic bead affinity column. miRNA-sequencing was performed on the isolated EVs, and although similar profiles were observed, a few key miRNAs involved in bone metabolism (miR-106b-5p, miRs-19b-3p, and miRs-219a-5p) were enriched in the Ocn-derived relative to the Prx1-derived EV subpopulations. To characterize the effects of these small EVs on a bone cell target, cultured mouse bone marrow stromal cells were treated with Prx1 or Ocn EVs, and mRNA-sequencing was performed. Pathways involved in ossification, bone development, and extracellular matrix interactions were regulated by both EV subpopulations, whereas a few pathways including advanced glycation end-products signaling were uniquely regulated in the Ocn EV subpopulation, underlying important biological effects of specific EV subpopulations within the bone marrow microenvironment. These data demonstrate that EV isolation in vivo using the CAGS-Snorkel mouse model is a useful tool in characterizing the cargo and understanding the biology of tissue-specific EVs. Moreover, while bone mesenchymal cell populations share a common EV secretory profile, we uncover key differences based on the stage of osteoblastic differentiation that may have important biological consequences.

Time-restricted feeding improves glucose homeostasis through epigenetic control of pancreatic β cell function.