Increased PTCHD4 expression via m6A modification of PTCHD4 mRNA promotes senescent cell survival.

RNA modifications, including N6-methyladenosine (m6A), critically modulate protein expression programs in a range of cellular processes. Although the transcriptomes of cells undergoing senescence are strongly regulated, the landscape and impact of m6A modifications during senescence are poorly understood. Here, we report a robust m6A modification of PTCHD4 mRNA, encoding Patched Domain-Containing Protein 4, in senescent cells. The METTL3/METTL14 complex was found to incorporate the m6A modification on PTCHD4 mRNA; addition of m6A rendered PTCHD4 mRNA more stable and increased PTCHD4 production. MeRIP RT-qPCR and eCLIP analyses were used to map this m6A modification to the last exon of PTCHD4 mRNA. Further investigation identified IGF2BP1, but not other m6A readers, as responsible for the stabilization and increased abundance of m6A-modified PTCHD4 mRNA. Silencing PTCHD4, a transmembrane protein, enhanced growth arrest and DNA damage in pre-senescent cells and sensitized them to senolysis and apoptosis. Our results indicate that m6A modification of PTCHD4 mRNA increases the production of PTCHD4, a protein associated with senescent cell survival, supporting the notion that regulating m6A modification on specific mRNAs could be exploited to eliminate senescent cells for therapeutic benefit.

Homeodomain-only protein suppresses proliferation and contributes to differentiation- and age-related reduced CD8+ T cell expansion.

T cell activation is a tightly controlled process involving both positive and negative regulators. The precise mechanisms governing the negative regulators in T cell proliferation remain incompletely understood. Here, we report that homeodomain-only protein (HOPX), a homeodomain-containing protein, and its most abundant isoform HOPXb, negatively regulate activation-induced proliferation of human T cells. We found that HOPX expression progressively increased from naïve (TN) to central memory (TCM) to effector memory (TEM) cells, with a notable upregulation following in vitro stimulation. Overexpression of HOPXb leads to a reduction in TN cell proliferation while HOPX knockdown promotes proliferation of TN and TEM cells. Furthermore, we demonstrated that HOPX binds to promoters and exerts repressive effects on the expression of MYC and NR4A1, two positive regulators known to promote T cell proliferation. Importantly, our findings suggest aging is associated with increased HOPX expression, and that knockdown of HOPX enhances the proliferation of CD8+ T cells in older adults. Our findings provide compelling evidence that HOPX serves as a negative regulator of T cell activation and plays a pivotal role in T cell differentiation and in age-related-reduction in T cell proliferation.

Transcriptional changes in the rat brain induced by repetitive transcranial magnetic stimulation.

Introduction: Transcranial Magnetic Stimulation (TMS) is a noninvasive technique that uses pulsed magnetic fields to affect the physiology of the brain and central nervous system. Repetitive TMS (rTMS) has been used to study and treat several neurological conditions, but its complex molecular basis is largely unexplored. Methods: Utilizing three experimental rat models (in vitro, ex vivo, and in vivo) and employing genome-wide microarray analysis, our study reveals the extensive impact of rTMS treatment on gene expression patterns. Results: These effects are observed across various stimulation protocols, in diverse tissues, and are influenced by time and age. Notably, rTMS-induced alterations in gene expression span a wide range of biological pathways, such as glutamatergic, GABAergic, and anti-inflammatory pathways, ion channels, myelination, mitochondrial energetics, multiple neuron-and synapse-specific genes. Discussion: This comprehensive transcriptional analysis induced by rTMS stimulation serves as a foundational characterization for subsequent experimental investigations and the exploration of potential clinical applications.

Sequential Early-Life Infections Alter Peripheral Blood Transcriptomics in Aging Female Mice but Not the Response to De Novo Infection with Influenza Virus or M. tuberculosis.

To determine the impact of accumulating Ag exposure on immunity in the aging mouse, and to develop a model more relevant to humans who are exposed to multiple pathogens during life, we sequentially infected young female mice with four distinct pathogens at 8-wk intervals: murine γ-herpesvirus 68, Sendai virus, murine CMV, and Heligmosomoides polygyrus. Mock-infected mice received PBS. After aging the sequentially infected and mock-infected mice to 18-25 mo under specific pathogen-free conditions, we analyzed multiple immune parameters. We assessed transcriptional activity in peripheral blood, T cell phenotype, the diversity of influenza epitopes recognized by CD8 T cells, and the response of the animals to infection with influenza virus and Mycobacterium tuberculosis. Our data show enhanced transcriptional activation in sequentially infected aged mice, with changes in some CD8 T cell subsets. However, there was no measurable difference in the response of mock-infected and sequentially infected aged mice to de novo infection with either influenza virus or M. tuberculosis at 18-21 mo. Unexpectedly, a single experiment in which 25-mo-old female mice were challenged with influenza virus revealed a significantly higher survival rate for sequentially infected (80%) versus mock-infected (20%) mice. These data suggest that although exposure to a variety of pathogen challenges in the mouse model does not overtly impact cellular markers of immunity in aged female mice following de novo respiratory infection, subtle changes may emerge in other compartments or with increasing age.

Cerebrospinal fluid exploratory proteomics and ketamine metabolite pharmacokinetics in human volunteers after ketamine infusion.

Ketamine is a treatment for both refractory depression and chronic pain syndromes. In order to explore ketamine's potential mechanism of action and whether ketamine or its metabolites cross the blood brain barrier, we examined the pharmacokinetics of ketamine and its metabolites-norketamine (NK), dehydronorketamine (DHNK), and hydroxynorketamines (HNKs)-in cerebrospinal fluid (CSF) and plasma, as well as in an exploratory proteomic analysis in the CSF of nine healthy volunteers who received ketamine intravenously (0.5 mg/kg IV). We found that ketamine, NK, and (2R,6R;2S,6S)-HNK readily crossed the blood brain barrier. Additionally, 354 proteins were altered in the CSF in at least two consecutive timepoints (p < 0.01). Proteins in the classes of tyrosine kinases, cellular adhesion molecules, and growth factors, including insulin, were most affected, suggesting an interplay of altered neurotransmission, neuroplasticity, neurogenesis, synaptogenesis, and neural network functions following ketamine administration.

Aberrant expression and localization of the RAP1 shelterin protein contribute to age-related phenotypes.

Short telomeres induce a DNA damage response (DDR) that evokes apoptosis and senescence in human cells. An extant question is the contribution of telomere dysfunction-induced DDR to the phenotypes observed in aging and telomere biology disorders. One candidate is RAP1, a telomere-associated protein that also controls transcription at extratelomeric regions. To distinguish these roles, we generated a knockin mouse carrying a mutated Rap1, which was incapable of binding telomeres and did not result in eroded telomeres or a DDR. Primary Rap1 knockin embryonic fibroblasts showed decreased RAP1 expression and re-localization away from telomeres, with an increased cytosolic distribution akin to that observed in human fibroblasts undergoing telomere erosion. Rap1 knockin mice were viable, but exhibited transcriptomic alterations, proinflammatory cytokine/chemokine signaling, reduced lifespan, and decreased healthspan with increased body weight/fasting blood glucose levels, spontaneous tumor incidence, and behavioral deficits. Taken together, our data present mechanisms distinct from telomere-induced DDR that underlie age-related phenotypes.

Early SRC activation skews cell fate from apoptosis to senescence.

Cells responding to DNA damage implement complex adaptive programs that often culminate in one of two distinct outcomes: apoptosis or senescence. To systematically identify factors driving each response, we analyzed human IMR-90 fibroblasts exposed to increasing doses of the genotoxin etoposide and identified SRC as a key kinase contributing early to this dichotomous decision. SRC was activated by low but not high levels of etoposide. With low DNA damage, SRC-mediated activation of p38 critically promoted expression of cell survival and senescence proteins, while SRC-mediated repression of p53 prevented a rise in proapoptotic proteins. With high DNA damage, failure to activate SRC led to elevation of p53, inhibition of p38, and apoptosis. In mice exposed to DNA damage, pharmacologic inhibition of SRC prevented the accumulation of senescent cells in tissues. We propose that inhibiting SRC could be exploited to favor apoptosis over senescence in tissues to improve health outcomes.

Loss of Elp1 perturbs histone H2A.Z and the Notch signaling pathway.

Elongator dysfunction is increasingly recognized as a contributor to multiple neurodevelopmental and neurodegenerative disorders including familial dysautonomia, intellectual disability, amyotrophic lateral sclerosis, and autism spectrum disorder. Although numerous cellular processes are perturbed in the context of Elongator loss, converging evidence from multiple studies has resolved Elongator's primary function in the cell to the modification of tRNA wobble uridines and the translational regulation of codon-biased genes. Here we characterize H2a.z, encoding the variant H2a histone H2A.Z, as an indirect Elongator target. We further show that canonical Notch signaling, a pathway directed by H2A.Z, is perturbed as a consequence of Elp1 loss. Finally, we demonstrate that hyperacetylation of H2A.Z and other histones via exposure to the histone deacetylase inhibitor Trichostatin A during neurogenesis corrects the expression of Notch3 and rescues the development of sensory neurons in embryos lacking the Elp1 Elongator subunit.

GRSF1 deficiency in skeletal muscle reduces endurance in aged mice.

GRSF1 is a mitochondrial RNA-binding protein important for maintaining mitochondrial function. We found that GRSF1 is highly expressed in cultured skeletal myoblasts differentiating into myotubes. To understand the physiological function of GRSF1 in vivo, we generated mice in which GRSF1 was specifically ablated in skeletal muscle. The conditional knockout mice (Grsf1cKO) appeared normal until 7-9 months of age. Importantly, however, a reduction of muscle endurance compared to wild-type controls was observed in 16- to 18-month old Grsf1cKO mice. Transcriptomic analysis revealed more than 200 mRNAs differentially expressed in Grsf1cKO muscle at this age. Notably, mRNAs encoding proteins involved in mitochondrial function, inflammation, and ion transport, including Mgarp, Cxcl10, Nfkb2, and Sln mRNAs, were significantly elevated in aged Grsf1cKO muscle. Our findings suggest that GRSF1 deficiency exacerbates the functional decline of aged skeletal muscle, likely through multiple downstream effector proteins.

Hepatocyte cannabinoid 1 receptor nullification alleviates toxin-induced liver damage via NF-κB signaling.

Cannabinoid 1 receptor (CB1R) expression is upregulated in the liver with viral hepatitis, cirrhosis, and both alcoholic and non-alcoholic fatty liver disease (FLD), whereas its expression is muted under usual physiological conditions. Inhibiting CB1R has been shown to be beneficial in preserving hepatic function in FLD but it is unclear if inhibiting CB1R during an inflammatory response to an acute hepatic injury, such as toxin-induced injury, would also be beneficial. We found that intrinsic CB1R in hepatocytes regulated liver inflammation-related gene transcription. We tested if nullification of hepatocyte-specific CB1R (hCNR1-/-) in mice protects against concanavalin A (Con A)-induced liver injury. We looked for evidence of liver damage and markers of inflammation in response to Con A by measuring liver enzyme levels and proinflammatory cytokines (e.g., TNF-α, IL-1ß, IL-6, IL-17) in serum collected from hCNR1-/- and control mice. We observed a shift to the right in the dose-response curve for liver injury and inflammation in hCNR1-/- mice. We also found less inflammatory cell infiltration and focal necrosis in livers of hCNR1-/- mice compared to controls, resulting from downregulated apoptotic markers. This anti-apoptotic mechanism results from increased activation of nuclear factor kappa B (NF-κB), especially cAMP-dependent cannabinoid signaling and membrane-bound TNF-α, via downregulated TNF-α receptor 2 (TNFR2) transcription levels. Collectively, these findings provide insight into involvement of CB1R in the pathogenesis of acute liver injury.

The association between poverty and gene expression within peripheral blood mononuclear cells in a diverse Baltimore City cohort.

Socioeconomic status (SES), living in poverty, and other social determinants of health contribute to health disparities in the United States. African American (AA) men living below poverty in Baltimore City have a higher incidence of mortality when compared to either white males or AA females living below poverty. Previous studies in our laboratory and elsewhere suggest that environmental conditions are associated with differential gene expression (DGE) patterns in peripheral blood mononuclear cells (PBMCs). DGE have also been associated with hypertension and cardiovascular disease (CVD) and correlate with race and sex. However, no studies have investigated how poverty status associates with DGE between male and female AAs and whites living in Baltimore City. We examined DGE in 52 AA and white participants of the Healthy Aging in Neighborhoods of Diversity across the Life Span (HANDLS) cohort, who were living above or below 125% of the 2004 federal poverty line at time of sample collection. We performed a microarray to assess DGE patterns in PBMCs from these participants. AA males and females living in poverty had the most genes differentially-expressed compared with above poverty controls. Gene ontology (GO) analysis identified unique and overlapping pathways related to the endosome, single-stranded RNA binding, long-chain fatty-acyl-CoA biosynthesis, toll-like receptor signaling, and others within AA males and females living in poverty and compared with their above poverty controls. We performed RT-qPCR to validate top differentially-expressed genes in AA males. We found that KLF6, DUSP2, RBM34, and CD19 are expressed at significantly lower levels in AA males in poverty and KCTD12 is higher compared to above poverty controls. This study serves as an additional link to better understand the gene expression response in peripheral blood mononuclear cells in those living in poverty.

Multidimensional informatic deconvolution defines gender-specific roles of hypothalamic GIT2 in aging trajectories.

In most species, females live longer than males. An understanding of this female longevity advantage will likely uncover novel anti-aging therapeutic targets. Here we investigated the transcriptomic responses in the hypothalamus - a key organ for somatic aging control - to the introduction of a simple aging-related molecular perturbation, i.e. GIT2 heterozygosity. Our previous work has demonstrated that GIT2 acts as a network controller of aging. A similar number of both total (1079-female, 1006-male) and gender-unique (577-female, 527-male) transcripts were significantly altered in response to GIT2 heterozygosity in early life-stage (2 month-old) mice. Despite a similar volume of transcriptomic disruption in females and males, a considerably stronger dataset coherency and functional annotation representation was observed for females. It was also evident that female mice possessed a greater resilience to pro-aging signaling pathways compared to males. Using a highly data-dependent natural language processing informatics pipeline, we identified novel functional data clusters that were connected by a coherent group of multifunctional transcripts. From these it was clear that females prioritized metabolic activity preservation compared to males to mitigate this pro-aging perturbation. These findings were corroborated by somatic metabolism analyses of living animals, demonstrating the efficacy of our new informatics pipeline.

Monoclonal Antibody to Marinobufagenin Downregulates TGFß Profibrotic Signaling in Left Ventricle and Kidney and Reduces Tissue Remodeling in Salt-Sensitive Hypertension.

Background Elevated levels of an endogenous Na/K-ATPase inhibitor marinobufagenin accompany salt-sensitive hypertension and are implicated in cardiac fibrosis. Immunoneutralization of marinobufagenin reduces blood pressure in Dahl salt-sensitive (Dahl-S) rats. The effect of the anti-marinobufagenin monoclonal antibody on blood pressure, left ventricular (LV) and renal remodeling, and gene expression were investigated in hypertensive Dahl-S rats. Methods and Results Dahl-S rats were fed high NaCl (8%, HS; n=14) or low NaCl (0.1%, LS; n=14) diets for 8 weeks. Animals were administered control antibody (LS control antibody, LSC; HS control antibody, HSC; n=7 per group) or anti-marinobufagenin antibody once on week 7 of diet intervention (n=7 per group). Levels of marinobufagenin, LV, and kidney mRNAs and proteins implicated in profibrotic signaling were assessed. Systolic blood pressure was elevated (211±8 versus 133±3 mm Hg, P<0.01), marinobufagenin increased 2-fold in plasma (P<0.05) and 5-fold in urine (P<0.01), LV and kidney weights increased, and levels of LV collagen-1 rose 3.5-fold in HSC versus LSC. Anti-marinobufagenin antibody treatment decreased systolic blood pressure by 24 mm Hg (P<0.01) and reduced organ weights and level of LV collagen-1 (P<0.01) in hypertensive Dahl salt-sensitive rats with anti-marinobufagenin antibody versus HSC. The expression of genes related to transforming growth factor-ß-dependent signaling was upregulated in the left ventricles and kidneys in HSC versus LSC groups and became downregulated following administration of anti-marinobufagenin antibody to hypertensive Dahl-S rats. Marinobufagenin also activated transforming growth factor-ß signaling in cultured ventricular myocytes from Dahl-S rats. Conclusions Immunoneutralization of heightened marinobufagenin levels in hypertensive Dahl-S rats resulted in a downregulation of genes implicated in transforming growth factor-ß pathway, which indicates that marinobufagenin is an activator of profibrotic transforming growth factor-ß-dependent signaling in salt-sensitive hypertension.

Loss of miR-451a enhances SPARC production during myogenesis.

MicroRNAs (miRNAs) are small noncoding RNAs that critically regulate gene expression. Their abundance and function have been linked to a range of physiologic and pathologic processes. In aged monkey muscle, miR-451a and miR-144-3p were far more abundant than in young monkey muscle. This observation led us to hypothesize that miR-451a and miR-144-3p may influence muscle homeostasis. To test if these conserved microRNAs were implicated in myogenesis, we investigated their function in the mouse myoblast line C2C12. The levels of both microRNAs declined with myogenesis; however, only overexpression of miR-451a, but not miR-144-3p, robustly impeded C2C12 differentiation, suggesting an inhibitory role for miR-451a in myogenesis. Further investigation of the regulatory influence of miR-451a identified as one of the major targets Sparc mRNA, which encodes a secreted protein acidic and rich in cysteine (SPARC) that functions in wound healing and cellular differentiation. In mouse myoblasts, miR-451a suppressed Sparc mRNA translation. Together, our findings indicate that miR-451a is downregulated in differentiated myoblasts and suggest that it decreases C2C12 differentiation at least in part by suppressing SPARC biosynthesis.

Muscle cannabinoid 1 receptor regulates Il-6 and myostatin expression, governing physical performance and whole-body metabolism.

Sarcopenic obesity, the combination of skeletal muscle mass and function loss with an increase in body fat, is associated with physical limitations, cardiovascular diseases, metabolic stress, and increased risk of mortality. Cannabinoid receptor type 1 (CB1R) plays a critical role in the regulation of whole-body energy metabolism because of its involvement in controlling appetite, fuel distribution, and utilization. Inhibition of CB1R improves insulin secretion and insulin sensitivity in pancreatic ß-cells and hepatocytes. We have now developed a skeletal muscle-specific CB1R-knockout (Skm-CB1R-/-) mouse to study the specific role of CB1R in muscle. Muscle-CB1R ablation prevented diet-induced and age-induced insulin resistance by increasing IR signaling. Moreover, muscle-CB1R ablation enhanced AKT signaling, reducing myostatin expression and increasing IL-6 secretion. Subsequently, muscle-CB1R ablation increased myogenesis through its action on MAPK-mediated myogenic gene expression. Consequently, Skm-CB1R-/- mice had increased muscle mass and whole-body lean/fat ratio in obesity and aging. Muscle-CB1R ablation improved mitochondrial performance, leading to increased whole-body muscle energy expenditure and improved physical endurance, with no change in body weight. These results collectively show that CB1R in muscle is sufficient to regulate whole-body metabolism and physical performance and is a novel target for the treatment of sarcopenic obesity. -González-Mariscal, I., Montoro, R. A., O'Connell, J. F., Kim, Y., Gonzalez-Freire, M., Liu, Q.-R., Alfaras, I., Carlson, O. D., Lehrmann, E., Zhang, Y., Becker, K. G., Hardivillé, S., Ghosh, P., Egan, J. M. Muscle cannabinoid 1 receptor regulates Il-6 and myostatin expression, governing physical performance and whole-body metabolism.

Topoisomerase 3ß interacts with RNAi machinery to promote heterochromatin formation and transcriptional silencing in Drosophila.

Topoisomerases solve topological problems during DNA metabolism, but whether they participate in RNA metabolism remains unclear. Top3ß represents a family of topoisomerases carrying activities for both DNA and RNA. Here we show that in Drosophila, Top3ß interacts biochemically and genetically with the RNAi-induced silencing complex (RISC) containing AGO2, p68 RNA helicase, and FMRP. Top3ß and RISC mutants are similarly defective in heterochromatin formation and transcriptional silencing by position-effect variegation assay. Moreover, both Top3ß and AGO2 mutants exhibit reduced levels of heterochromatin protein HP1 in heterochromatin. Furthermore, expression of several genes and transposable elements in heterochromatin is increased in the Top3ß mutant. Notably, Top3ß mutants defective in either RNA binding or catalytic activity are deficient in promoting HP1 recruitment and silencing of transposable elements. Our data suggest that Top3ß may act as an RNA topoisomerase in siRNA-guided heterochromatin formation and transcriptional silencing.

Hydroxyurea attenuates oxidative, metabolic, and excitotoxic stress in rat hippocampal neurons and improves spatial memory in a mouse model of Alzheimer's disease.

Alzheimer's disease (AD) is an age-related neurodegenerative disorder characterized by accumulation of amyloid ß-peptide (Aß) plaques in the brain and decreased cognitive function leading to dementia. We tested if hydroxyurea (HU), a ribonucleotide reductase inhibitor known to activate adaptive cellular stress responses and ameliorate abnormalities associated with several genetic disorders, could protect rat hippocampal neurons against oxidative-, excitatory-, mitochondrial-, and Aß-induced stress and if HU treatment could improve learning and memory in the APP/PS1 mouse model of AD. HU treatment attenuated the loss of cell viability induced by treatment of hippocampal neurons with hydrogen peroxide, glutamate, rotenone, and Aß1-42. HU treatment attenuated reductions of mitochondrial reserve capacity, maximal respiration, and cellular adenosine triphosphate content induced by hydrogen peroxide treatment. In vivo, treatment of APP/PS1 mice with HU (45 mg/kg/d) improved spatial memory performance in the hippocampus-dependent Morris water maze task without reducing Aß levels. HU provides neuroprotection against toxic insults including Aß, improves mitochondrial bioenergetics, and improves spatial memory in an AD mouse model. HU may offer a new therapeutic approach to delay cognitive decline in AD.

MIR100 host gene-encoded lncRNAs regulate cell cycle by modulating the interaction between HuR and its target mRNAs.

Long non-coding RNAs (lncRNAs) regulate vital biological processes, including cell proliferation, differentiation and development. A subclass of lncRNAs is synthesized from microRNA (miRNA) host genes (MIRHGs) due to pre-miRNA processing, and are categorized as miRNA-host gene lncRNAs (lnc-miRHGs). Presently, the cellular function of most lnc-miRHGs is not well understood. We demonstrate a miRNA-independent role for a nuclear-enriched lnc-miRHG in cell cycle progression. MIR100HG produces spliced and stable lncRNAs that display elevated levels during the G1 phase of the cell cycle. Depletion of MIR100HG-encoded lncRNAs in human cells results in aberrant cell cycle progression without altering the levels of miRNA encoded within MIR100HG. Notably, MIR100HG interacts with HuR/ELAVL1 as well as with several HuR-target mRNAs. Further, MIR100HG-depleted cells show reduced interaction between HuR and three of its target mRNAs, indicating that MIR100HG facilitates interaction between HuR and target mRNAs. Our studies have unearthed novel roles played by a MIRHG-encoded lncRNA in regulating RNA binding protein activity, thereby underscoring the importance of determining the function of several hundreds of lnc-miRHGs that are present in human genome.

Eda-activated RelB recruits an SWI/SNF (BAF) chromatin-remodeling complex and initiates gene transcription in skin appendage formation.

Ectodysplasin A (Eda) signaling activates NF-κB during skin appendage formation, but how Eda controls specific gene transcription remains unclear. Here, we find that Eda triggers the formation of an NF-κB-associated SWI/SNF (BAF) complex in which p50/RelB recruits a linker protein, Tfg, that interacts with BAF45d in the BAF complex. We further reveal that Tfg is initially induced by Eda-mediated RelB activation and then bridges RelB and BAF for subsequent gene regulation. The BAF component BAF250a is particularly up-regulated in skin appendages, and epidermal knockout of BAF250a impairs skin appendage development, resulting in phenotypes similar to those of Eda-deficient mouse models. Transcription profiling identifies several target genes regulated by Eda, RelB, and BAF. Notably, RelB and the BAF complex are indispensable for transcription of Eda target genes, and both BAF complex and Eda signaling are required to open chromatin of Eda targets. Our studies thus suggest that Eda initiates a signaling cascade and recruits a BAF complex to specific gene loci to facilitate transcription during organogenesis.

Altered Extracellular Vesicle Concentration, Cargo, and Function in Diabetes.

Type 2 diabetes is a chronic age-associated degenerative metabolic disease that reflects relative insulin deficiency and resistance. Extracellular vesicles (EVs) (exosomes, microvesicles, and apoptotic bodies) are small (30-400 nm) lipid-bound vesicles capable of shuttling functional proteins, nucleic acids, and lipids as part of intercellular communication systems. Recent studies in mouse models and in cell culture suggest that EVs may modulate insulin signaling. Here, we designed cross-sectional and longitudinal cohorts of euglycemic participants and participants with prediabetes or diabetes. Individuals with diabetes had significantly higher levels of EVs in their circulation than euglycemic control participants. Using a cell-specific EV assay, we identified that levels of erythrocyte-derived EVs are higher with diabetes. We found that insulin resistance increases EV secretion. Furthermore, the levels of insulin signaling proteins were altered in EVs from individuals with high levels of insulin resistance and ß-cell dysfunction. Moreover, EVs from individuals with diabetes were preferentially internalized by circulating leukocytes. Cytokine levels in the media and in EVs were higher from monocytes incubated with diabetic EVs. Microarray of these leukocytes revealed altered gene expression pathways related to cell survival, oxidative stress, and immune function. Collectively, these results suggest that insulin resistance increases the secretion of EVs, which are preferentially internalized by leukocytes, and alters leukocyte function.