Proinflammatory Stress Activates Neutral Sphingomyelinase 2-Based Generation of a Ceramide-Enriched β-Cell EV Subpopulation

Abstract

β-Cell extracellular vesicles (EVs) play a role as paracrine effectors in islet health, yet mechanisms connecting β-cell stress to changes in EV cargo and potential impacts on diabetes remain poorly defined. We hypothesized that β-cell inflammatory stress engages neutral sphingomyelinase 2 (nSMase2)-dependent EV formation pathways, generating ceramide-enriched small EVs that could impact surrounding β-cells. Consistent with this, proinflammatory cytokine treatment of INS-1 β-cells and human islets concurrently increased β-cell nSMase2 and ceramide abundance, as well as small EV ceramide species. Direct chemical activation or genetic knockdown of nSMase2, chemical treatment to inhibit cell death pathways, or treatment with a glucagon-like peptide-1 (GLP-1) receptor agonist also modulated β-cell EV ceramide. RNA sequencing of ceramide-enriched EVs identified a distinct set of miRNAs linked to β-cell function and identity. EV treatment from cytokine-exposed parent cells inhibited peak glucose-stimulated insulin secretion in wild-type recipient cells; this effect was abrogated when using EVs from nSMase2 knockdown parent cells. Finally, plasma EVs in children with recent-onset type 1 diabetes showed increases in multiple ceramide species. These findings highlight nSMase2 as a regulator of β-cell EV cargo and identify ceramide-enriched EV populations as a contributor to EV-related paracrine signaling under conditions of β-cell inflammatory stress and death.

Article highlights: Mechanisms connecting β-cell stress to extracellular vesicle (EV) cargo and diabetes are poorly defined. Does β-cell inflammatory stress engage neutral sphingomyelinase 2 (nSMase2)-dependent EV formation to generate ceramide-enriched small EVs? Proinflammatory cytokines increased β-cell small EV ceramide via increases in nSMase2. Ceramide-enriched EVs housed distinct cargo linked to insulin signaling, and ceramide species were enriched in plasma EVs from individuals with type 1 diabetes. Ceramide-enriched EV populations are a potential contributor to β-cell EV-related paracrine signaling.

Figure 1

β-Cell nSMase2 expression and ceramide production are increased in concert under conditions of T1D and cytokine exposure. A: Violin plots of β-cell Smpd3 expression in control, T1D, and T2D models show a significant upregulation in β-cells from the NOD mouse model of T1D (P < 0.01). B–E: In INS-1 β-cells, 24-h IL-1β increases nSMase2 as measured via immunoblot (B) (with quantification as shown) or flow cytometry staining (C), as well as total cellular ceramides (D) and surface ceramide flow cytometry staining (E). INS-1 experiments were performed using 3–11 biological replicates, as plotted. Data in C and E were compared using Student t tests, and data in D were compared using Kruskal-Wallis ANOVA with Tukey adjustment for multiple comparisons. F and G: Human islets were treated with a 48-h mix of IL-1β (5 ng/mL), TNF-α (10 ng/mL), and IFN-γ (100 ng/mL), then dispersed for flow cytometry–based quantification of nSMase2 (F) and ceramides (G). Data are shown for five unique donors for whom samples were tested in triplicate, and changes were compared using a Student t test. Significant differences were also present when using a paired t test to compare mean donor values between control and untreated islets (n = 5 per group). Data are mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. Cyto, cytokine mix; MFI, mean fluorescence intensity; Veh, vehicle.

Figure 2

In concert with cellular nSMase2 and ceramide, cytokine treatment increases β-cell EV ceramides. A and B: β-Cell small EVs were isolated using bead-based tetraspanin antibody pulldown, eluted, washed, concentrated, and verified using TEM (direct magnification ×110,000) (A) and NTA (B). C and D: Flow cytometry–based staining for ceramide was performed to assess EV ceramide content in control and cytokine-treated INS-1 cells (C) and human islets (D). INS-1 experiments were performed using 12 biological replicates, as plotted, and compared using Kruskal-Wallis ANOVA with Tukey multiple comparisons test. Human islet samples were tested in triplicate, and mean values for 10 unique donors were compared using a paired t test. Data are mean ± SEM. **P < 0.01, ***P < 0.001. Cyto, cytokine mix; MFI, mean fluorescence intensity; Veh, vehicle.

Figure 3

Modulation of nSMase2 activity impacts β-cell ceramide-enriched EV generation. A–C: INS-1 β-cells were treated with 24 h of CAPE (5 µmol/L), which increased cell ceramides (A), total EV secretion (B), and EV ceramide staining (C) with or without concurrent cytokine treatment. D: Stable nSMase2 KD INS-1 β-cells were developed using rat nSMase2 shRNA to reduce cellular nSMase2 levels at baseline and in response to cytokines (10 ng/mL IL-1β). E: nSMase2 KD also reduced cytokine-induced increases in cellular ceramide staining. F and G: nSMase2 KD reduced total EV secretion and drastically reduced EV ceramide. H: Raptinal treatment to directly induce apoptosis increased EV ceramide content in mouse islets. I: ZVAD treatment to inhibit caspase-induced cell death abrogated cytokine-induced increases in small EV ceramide. J: Treatment with 5 µmol/L GSK-872 for 24 h to inhibit RIPK3-induced cell death also abrogated cytokine-induced increases in small EV ceramide. K–M: INS-1 β-cells were treated with the diabetes therapeutic and GLP-1 receptor agonist exendin-4 at 20 nmol/L for 24 h. Exendin-4 treatment significantly abrogated IL-1β–induced cellular nSMase2 (K) and cellular ceramide staining (L). Exendin-4 also significantly decreased EV ceramide staining (M). Data in A, I, and J were compared using one-way ANOVA with Tukey multiple comparisons test, while a nonparametric ANOVA with Dunn multiple comparisons test was applied to K–M. Two-way ANOVA with Tukey multiple comparisons test was used to compare data in D and E. Data in B were tested with a Student t test, while C, F, G, and M were compared using a nonparametric Mann-Whitney test. Data are mean ± SEM (n = 3 biological replicates for NTA experiments and nSMase2 KD validation and 6–12 biological replicates for other panels, as plotted). *P < 0.05, **P < 0.01, ***P < 0.001. EX, exendin-4; MFI, mean fluorescence intensity; SCR, scramble.

Figure 4

Tunicamycin, thapsigargin, and doxorubicin treatment increase β-cell nSMase2 and ceramide production in INS-1 cells. A–I: In INS-1 cells, 24-h exposure to tunicamycin (1 µmol/L) (A–C), thapsigargin (5 nmol/L) (D–F), or doxorubicin (50 nmol/L) (G–I) increased nSMase2 (A, D, and G) and ceramides (B, E, and H), as well as ceramides in EVs (C, F, and I). EVs were isolated by immunoaffinity using tetraspanin antibodies. Ceramide content on EVs was determined by flow cytometry using ceramide antibody. INS-1 experiments were performed using 3–12 biological replicates, as plotted, and compared using Student t test (A–D, and I) or nonparametric Mann-Whitney test (E–H). Data are mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. Doxo, doxorubicin; MFI, mean fluorescence intensity; TG, thapsigargin; TUN, tunicamycin.

Figure 5

β-Cell cytokine-induced ceramide-enriched EV subpopulations impact recipient cell GSIS and carry differential miRNA cargo compared with global EV populations. A and B: INS-1 scramble control (A) or nSMase2 KD cells (B) were exposed to IL-1β or vehicle control for 24 h. Small EVs were isolated from each well of parent cells, then used for treatment of wild-type recipient INS cells at a 5 × 10⁸/mL concentration for 48 h. GSIS was measured in wild-type cells. Three biological replicates per group were compared using two-way ANOVA with Tukey posttest. C and D: Wild-type INS-1 cells were exposed to IL-1β or vehicle control for 24 h. Ceramide-enriched EVs and tetraspanin pulldown EVs were isolated using streptavidin magnetic beads and biotinylated antibodies against ceramide or tetraspanins. EV RNA was isolated and subjected to miRNA sequencing. C: One-dimensional plot of multidimensional scaling on miRNA showed baseline differences in miRNA expression profiles between ceramide-enriched EVs and global EVs. D: The highest scored network was generated using QIAGEN IPA with a set of cutoffs (down and up 1.5-fold of expression fold change (FC), P < 0.05 and FDR < 0.05). The network suggested that differential miRNAs were involved in a series of network nodes, including insulin. dim, dimension; HG, high glucose; LG, low glucose; Scr, scramble; Veh, vehicle.