Biology subjects

Kulkarni, R. N.

Publications and source records attributed to Kulkarni, R. N..

13 recordsLinked to original sources

bioRxiv2025

Single-Islet Proteomics Maps Pseudo-Temporal Islet Immune Responses and Dysfunction in Stage 1 Type 1 Diabetes

Progressive {beta}-cell dysfunction precedes the onset of type 1 diabetes (T1D), yet the molecular mechanisms driving early T1D development remain poorly understood. Although single-cell RNA-sequencing has uncovered transcript-level changes in human islet cells, it offers limited insight into the heterogeneity of distinct islet microenvironments. Here, we applied a single-islet proteomics workflow to profile intra-donor islet heterogeneity in three stage 1 T1D cases with matched non-diabetic controls and define in situ protein signatures of pseudo-temporal islet dysfunction. Intra-donor analyses of [~]100 individual islets per donor revealed highly consistent proteomic patterns reflecting pseudo-time progression of islet immune responses and {beta}-cell dysfunction. Several pathways, including extracellular matrix remodeling and mRNA processing, were identified as closely associated with progressive islet immune activation and loss of {beta}-cell function. These findings provide robust proteome-wide evidence of the progression of islet dysfunction, offer a valuable resource for investigating early mechanisms of T1D pathogenesis-- including novel candidates for functional studies--and underscore the utility of single-islet spatial proteomics for examining islet heterogeneity in T1D.

systems biology↗

bioRxiv2025

Exploring Size Exclusion Chromatography Columns 20 and 35 nm Pore Size Effect for Isolation of Extracellular Vesicles

Extracellular vesicles (EVs) have shown great promise as minimally invasive biomarkers for a variety of diseases. However, challenges persist regarding EV isolation, particularly in their co-isolation with impurities such as soluble proteins and lipoproteins. Among the methods available for EV isolation, size-exclusion chromatography (SEC) is widely used, as it is reproducible and amenable to high-throughput with a rapid turnaround time. However, its size-based separation leads to the co-isolation of EVs with impurities of similar size. This study, for the first time to our knowledge, compares SEC columns with different pore sizes, 20 and 35 nm, to evaluate their efficacy in non-EV contaminant removal and EV recovery from pancreatic EndoC-{beta}H1 cell culture media and human plasma. To assess EV purity and yield, we compare EV particle concentration, the presence of unintended co-isolates, and RNA EV cargo. This study demonstrates that smaller pore size SEC columns enhance EV yield and purity, making them ideal for biomarker studies involving limited biological samples or downstream analysis sensitive to contaminants.

biochemistry↗

bioRxiv2025

Walking speed can be modulated on an adaptive split-belt treadmill

Adaptive treadmills (ATMs) that change speed based on the users gait mechanics can allow for healthy stride-to-stride variability observed during overground walking, while maintaining the benefits of traditional treadmill training for gait rehabilitation. To enable unilateral targeting of propulsion, we developed an adaptive split-belt treadmill (sATM) that updates the speed of each belt of the treadmill based on the users propulsion and position. The present study demonstrated equivalence of the sATM to an existing tied-belt ATM that updates the speed of both belts based on the users propulsion, step length, and position. Fourteen young, healthy participants completed five trials at their comfortable walking speed on the tied ATM and sATM with unilateral and bilateral modifications. We hypothesized that propulsion and belt speed on the novel sATM would be equivalent to the tied ATM, and that the sATM could be modified to encourage higher propulsion unilaterally while maintaining walking speed. Participants maintained similar propulsion and belt speeds between treadmills and belts for bilateral modifications. While some participants increased unilateral propulsion to maintain similar walking speeds for the unilateral modification, others showed no change in propulsion with slight differences in walking speed. Our results suggest that the response to unilateral modifications was user-specific, and the sATM could be tuned individually to allow unilateral control strategies. The present study demonstrates that walking speed can be modulated on an adaptive split-belt treadmill.

bioengineering↗

bioRxiv2025

Characterization of cytokine treatment on human pancreatic islets by top-down proteomics

Type 1 diabetes (T1D) results from autoimmune-mediated destruction of insulin-producing {beta} cells in the pancreatic islet. This process is modulated by pro-inflammatory cytokine signaling, which have been previously shown to alter protein expression in ex vivo islets. Herein, we applied top-down proteomics to globally evaluate proteoforms from human islets treated with proinflammatory cytokines (interferon-{gamma} and interleukin-1{beta}). We measured 1636 unique proteoforms across 6 donors and two time points (control and 24-hours post-treatment) and observed consistent changes in abundance across the glicentin-related pancreatic polypeptide (GRPP) and major proglucagon fragment regions of glucagon, as well as the LF-19/catestatin and vasostatin-1/2 region of chromogranin-A. We also observe several proteoforms that increase after cytokine-treatment or are exclusively observed after cytokine-treatment including forms of beta-2 Microglobulin (B2M), high-mobility group N2 protein (HMGN2), and chemokine (C-X-C motif) ligands (CXCL). Together, our quantitative results provide a baseline proteoform profile for human islets and identify several proteoforms that may serve as interesting candidate markers for T1D progression or therapeutic intervention. Statement of significance of the studyThis work applies a top-down proteomics workflow for the characterization and label-free quantification of proteoforms from human islets in the context of inflammation. The workflow is optimized for challenges unique to the islet proteome including high disulfide-linkage content and frequent truncation events, resulting in many proteoforms <5 kDa. There are limited examples of top-down proteomics characterization of human islets, thus this study provides a baseline characterization of the proteoforms of major hormones including insulin, glucagon, chromogranin-A and -B, and somatostatin. The quantitative results of proteoform abundances before and after cytokine treatment, which mimics the proinflammatory environment during T1D progression, enables the discovery of interesting proteoform candidates as potential markers of T1D progression.

biochemistry↗

bioRxiv2024

Increased Inflammation as well as Decreased Endoplasmic Reticulum Stress and Translation Differentiate Pancreatic Islets of Pre-symptomatic Stage 1 Type 1 Diabetes and Non-diabetic Cases

Aims/hypothesisProgression to type 1 diabetes (T1D) is associated with genetic factors, the presence of autoantibodies, and a decline in {beta} cell insulin secretion in response to glucose. Very little is known regarding the molecular changes that occur in human insulin-secreting {beta}-cells prior to the onset of T1D. Herein, we applied an unbiased proteomics approach to identify changes in proteins and potential mechanisms of islet dysfunction in islet autoantibody-positive organ donors with pre-symptomatic stage 1 T1D (HbA1c [≤] 6). We aimed to identify pathways in islets that are indicative of {beta}-cell dysfunction. MethodsMultiple islet sections were collected through laser microdissection of frozen pancreatic tissues of organ donors positive for islet autoantibodies (AAb+, n=5), compared to age/sex-matched nondiabetic controls (ND, n=5) obtained from the Network for Pancreatic Organ donors with Diabetes (nPOD). Islet sections were subjected to mass spectrometry-based proteomics and analyzed with label-free quantification followed by pathway and functional annotations. ResultsAnalyses resulted in [~]4,500 proteins identified with low false discovery rate (FDR) <1%, with 2,165 proteins reliably quantified in every islet sample. We observed large inter-donor variations that presented a challenge for statistical analysis of proteome changes between donor groups. We therefore focused on the three multiple AAb+ cases (mAAb+) with high genetic risk and their three matched controls for a final statistical analysis. Approximately 10% of the proteins (n=202) were significantly different between mAAb+ cases versus ND. The significant alterations clustered around major functions for upregulation in the immune response and glycolysis, and downregulation in endoplasmic reticulum (ER) stress response as well as protein translation and synthesis. The observed proteome changes were further supported by several independent published datasets, including proteomics dataset from in vitro proinflammatory cytokine-treated human islets and single cell RNA-seq data sets from AAb+ cases. Conclusion/interpretationIn-situ human islet proteome alterations at the stage 1 of AAb+ T1D centered around several major functional categories, including an expected increase in immune response genes (elevated antigen presentation / HLA), with decreases in protein synthesis and ER stress response, as well as compensatory metabolic response. The dataset serves as a proteomics resource for future studies on {beta} cell changes during T1D progression and pathogenesis.

pathology↗

bioRxiv2023

m6A mRNA Methylation Regulates Early Pancreatic β-Cell Differentiation

N6-methyladenosine (m6A) is the most abundant chemical modification in mRNA, and plays important roles in human and mouse embryonic stem cell pluripotency, maintenance, and differentiation. We have recently reported, for the first time, the role of m6A in the postnatal control of {beta}-cell function in physiological states and in Type 1 and 2 Diabetes. However, the precise mechanisms by which m6A acts to regulate the development of human and mouse {beta}-cells are unexplored. Here, we show that the m6A landscape is dynamic during human pancreas development, and that METTL14, one of the m6A writer complex proteins, is essential for the early differentiation of both human and mouse {beta}-cells.

developmental biology↗

bioRxiv2023

Stability and Manoeuvrability Interactions During Human Walking Depend on the Manoeuvre Direction

People use the mechanical interplay between stability and manoeuvrability to successfully walk. During single limb support, body states (position and velocity) that increase lateral stability will inherently resist lateral manoeuvres, decrease medial stability, and facilitate medial manoeuvres. Although not well understood, people can make behavioural decisions exploiting this relationship in anticipation of perturbations or direction changes. To characterize the behavioural component of the stability-manoeuvrability relationship, twenty-four participants performed many repetitions of a discrete stepping task involving mid-trial reactive manoeuvres (medial or lateral direction) in a Baseline (no external perturbations) and Perturbed (random mediolateral perturbations applied to their pelvis) environment. We hypothesized people would make systematic changes in lateral stability dependent on both environment (increasing lateral stability in the Perturbed environment) and anticipated manoeuvre direction (reducing lateral stability to facilitate lateral manoeuvres). Participants increased lateral stability in the Perturbed environment, coinciding with an increase in manoeuvre reaction time for laterally but not medially directed manoeuvres. Moreover, we observed lower lateral stability in both environments when people anticipated making a lateral manoeuvre when compared to medial manoeuvres. These results support the hypothesis that people behaviourally exploit the mechanical relationship between lateral stability and manoeuvrability depending on walk task goals and external environment.

bioengineering↗

bioRxiv2023

m6A mRNA Methylation in Brown Adipose Tissue Regulates Systemic Insulin Sensitivity via an Inter-Organ Prostaglandin Signaling Axis

Brown adipose tissue (BAT) has the capacity to regulate systemic metabolism through the secretion of signaling lipids. N6-methyladenosine (m6A) is the most prevalent and abundant post-transcriptional mRNA modification and has been reported to regulate BAT adipogenesis and energy expenditure. In this study, we demonstrate that the absence of m6A methyltransferase-like 14 (METTL14), modifies the BAT secretome to initiate inter-organ communication to improve systemic insulin sensitivity. Importantly, these phenotypes are independent of UCP1-mediated energy expenditure and thermogenesis. Using lipidomics, we identified prostaglandin E2 (PGE2) and prostaglandin F2a (PGF2a) as M14KO-BAT-secreted insulin sensitizers. Notably, circulatory PGE2 and PGF2a levels are inversely correlated with insulin sensitivity in humans. Furthermore, in vivo administration of PGE2 and PGF2a in high-fat diet-induced insulin-resistant obese mice recapitulates the phenotypes of METTL14 deficient animals. PGE2 or PGF2a improves insulin signaling by suppressing the expression of specific AKT phosphatases. Mechanistically, METTL14-mediated m6A installation promotes decay of transcripts encoding prostaglandin synthases and their regulators in human and mouse brown adipocytes in a YTHDF2/3-dependent manner. Taken together, these findings reveal a novel biological mechanism through which m6A-dependent regulation of BAT secretome regulates systemic insulin sensitivity in mice and humans. HighlightsO_LIMettl14KO-BAT improves systemic insulin sensitivity via inter-organ communication; C_LIO_LIPGE2 and PGF2a are BAT-secreted insulin sensitizers and browning inducers; C_LIO_LIPGE2 and PGF2a sensitize insulin responses through PGE2-EP-pAKT and PGF2a-FP-AKT axis; C_LIO_LIMETTL14-mediated m6A installation selectively destabilizes prostaglandin synthases and their regulator transcripts; C_LIO_LITargeting METTL14 in BAT has therapeutic potential to enhance systemic insulin sensitivity C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=139 SRC="FIGDIR/small/542169v1_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@16b3393org.highwire.dtl.DTLVardef@1072ae3org.highwire.dtl.DTLVardef@190a6d7org.highwire.dtl.DTLVardef@1b98bce_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2023

Redox Regulation of m6A Methyltransferase METTL3 in Human β-cells Controls the Innate Immune Response in Type 1 Diabetes

Type 1 Diabetes (T1D) is characterized by autoimmune-mediated destruction of insulin-producing {beta}-cells. Several observations have renewed interest in the innate immune system as an initiator of the disease process against {beta}-cells. Here, we show that N6-Methyladenosine (m6A) is an adaptive {beta}-cell safeguard mechanism that accelerates mRNA decay of the 2-5-oligoadenylate synthetase (OAS) genes to control the antiviral innate immune response at T1D onset. m6A writer methyltransferase 3 (METTL3) levels increase drastically in human and mouse {beta}-cells at T1D onset but rapidly decline with disease progression. Treatment of human islets and EndoC-{beta}H1 cells with pro-inflammatory cytokines interleukin-1 {beta} and interferon mimicked the METTL3 upregulation seen at T1D onset. Furthermore, m6A-sequencing revealed the m6A hypermethylation of several key innate immune mediators including OAS1, OAS2, and OAS3 in human islets and EndoC-{beta}H1 cells challenged with cytokines. METTL3 silencing in human pseudoislets or EndoC-{beta}H1 cells enhanced OAS levels by increasing its mRNA stability upon cytokine challenge. Consistently, in vivo gene therapy, to prolong Mettl3 overexpression specifically in {beta}-cells, delayed diabetes progression in the non-obese diabetic (NOD) mouse model of T1D by limiting the upregulation of Oas pointing to potential therapeutic relevance. Mechanistically, the accumulation of reactive oxygen species blocked METTL3 upregulation in response to cytokines, while physiological levels of nitric oxide promoted its expression in human islets. Furthermore, for the first time to our knowledge, we show that the cysteines in position C276 and C326 in the zinc finger domain of the METTL3 protein are sensitive to S-nitrosylation (SNO) and are significant for the METTL3 mediated regulation of OAS mRNA stability in human {beta}-cells in response to cytokines. Collectively, we report that m6A regulates human and mouse {beta}-cells to control the innate immune response during the onset of T1D and propose targeting METTL3 to prevent {beta}-cell death in T1D.

molecular biology↗

bioRxiv2023

Fructose Induced KHK-C Increases ER Stress and Modulates Hepatic Transcriptome to Drive Liver Disease in Diet-Induced and Genetic Models of NAFLD.

Non-alcoholic fatty liver disease (NAFLD) is a liver manifestation of metabolic syndrome, and is estimated to affect one billion individuals worldwide. An increased intake of a high-fat diet (HFD) and sugar-sweetened beverages are risk-factors for NAFLD development, but how their combined intake promotes progression to a more severe form of liver injury is unknown. Here we show that fructose metabolism via ketohexokinase (KHK) C isoform increases endoplasmic reticulum (ER) stress in a dose dependent fashion, so when fructose is coupled with a HFD intake it leads to unresolved ER stress. Conversely, a liver-specific knockdown of KHK in C57BL/6J male mice consuming fructose on a HFD is adequate to improve the NAFLD activity score and exert a profound effect on the hepatic transcriptome. Overexpression of KHK-C in cultured hepatocytes is sufficient to induce ER stress in fructose free media. Upregulation of KHK-C is also observed in genetically obesity ob/ob, db/db and lipodystrophic FIRKO male mice, whereas KHK knockdown in these mice improves metabolic function. Additionally, in over 100 inbred strains of male or female mice hepatic KHK expression correlates positively with adiposity, insulin resistance, and liver triglycerides. Similarly, in 241 human subjects and their controls, hepatic Khk expression is upregulated in early, but not late stages of NAFLD. In summary, we describe a novel role of KHK-C in triggering ER stress, which offers a mechanistic understanding of how the combined intake of fructose and a HFD propagates the development of metabolic complications.

molecular biology↗

bioRxiv2022

Fluorescein-based sensors to purify human α-cells for functional and transcriptomic analyses

Pancreatic -cells secrete glucagon, an insulin counter-regulatory peptide hormone critical for the maintenance of glucose homeostasis. Investigation of the function of human -cells remains a challenge due to the lack of cost-effective purification methods to isolate high-quality -cells from islets. Here, we use the reaction-based probe diacetylated Zinpyr1 (DA-ZP1) to introduce a novel and simple method for enriching live -cells from dissociated human islet cells with > 97% purity. The -cells, confirmed by sorting and immunostaining for glucagon, were cultured up to 10 days to form -pseudoislets. The -pseudoislets could be maintained in culture without significant loss of viability, and responded to glucose challenge by secreting appropriate levels of glucagon. RNA-sequencing analyses (RNA-seq) revealed that expression levels of key -cell identity genes were sustained in culture while some of the genes such as DLK1, GSN, SMIM24 were altered in -pseudoislets in a time-dependent manner. In conclusion, we report a method to sort human primary -cells with high purity that can be used for downstream analyses such as functional and transcriptional studies.

cell biology↗

bioRxiv2021

ROCK1 regulates insulin secretion from β-cells

ObjectiveThe endocrine pancreatic {beta}-cells play a pivotal role in the maintenance of whole-body glucose homeostasis and its dysregulation is a consistent feature in all forms of diabetes. However, knowledge of intracellular regulators that modulate {beta}-cell function remains incomplete. We investigated the physiological role of ROCK1 in the regulation of insulin secretion and glucose homoeostasis. MethodsMice lacking ROCK1 in pancreatic {beta}-cells (RIP-Cre; ROCK1loxP/loxP, {beta}-ROCK1-/-) were studied. Glucose and insulin tolerance tests as well as glucose-stimulated insulin secretion (GSIS) were measured. Insulin secretion response to a direct glucose or pyruvate or pyruvate kinase (PK) activator stimulation in isolated islets from {beta}-ROCK1-/- mice or {beta}-cell lines with knockdown of ROCK1 were also evaluated. Proximity ligation assay was performed to determine the physical interactions between PK and ROCK1. ResultsMice with a deficiency of ROCK1 in pancreatic {beta}-cells exhibited significantly increased blood glucose levels and reduced serum insulin without changes in body weight. Interestingly, {beta}-ROCK1-/- mice displayed progressive impairment of glucose tolerance while maintaining insulin sensitivity mostly due to impaired GSIS. Consistently, GSIS was markedly decreased in ROCK1-deficient islets and ROCK1 knockdown INS-1 cells. Concurrently, ROCK1 blockade led to a significant decrease in intracellular calcium levels, ATP levels, and oxygen consumption rates in isolated islets and INS-1 cells. Treatment of ROCK1-deficient islets or ROCK1 knockdown {beta}-cells either with pyruvate or a PK activator rescued the impaired GSIS. Mechanistically, we observed that ROCK1 binding to PK is greatly enhanced by glucose stimulation in {beta}-cells. ConclusionsOur findings demonstrate that {beta}-cell ROCK1 is essential for glucose-stimulated insulin secretion and maintenance of glucose homeostasis and that ROCK1 acts as an upstream regulator of glycolytic pyruvate kinase signaling.

physiology↗

bioRxiv2019

RADAR: Differential analysis of MeRIP-seq data with a random effect model

Epitranscriptome profiling using MeRIP-seq is a powerful technique for in vivo functional studies of reversible RNA modifications. We develop RADAR, a comprehensive analytical tool for detecting differentially methylated loci in MeRIP-seq data. RADAR enables accurate identification of altered methylation sites by accommodating variability of pre-immunoprecipitation expression level and post-immunoprecipitation count using different strategies. In addition, it is compatible with complex study design when covariates need to be incorporated in the analysis. Through simulation and real datasets analyses, we show that RADAR leads to more accurate and reproducible differential methylation analysis results than alternatives, which is available at https://github.com/scottzijiezhang/RADAR.

bioinformatics↗