Insulin Peptide Research Articles

Effects of Digitization of Self-Monitoring of Blood Glucose Records Using a Mobile App and the Cloud System on Outpatient Management of Diabetes: Single-Armed Prospective Study.

In recent years, technologies promoting the digitization of self-monitoring of blood glucose (SMBG) records including app-cloud cooperation systems have emerged. Studies combining these technological interventions with support from remote health care professionals have reported improvements in glycemic control. To assess the use of an app-cloud cooperation system linked with SMBG devices in clinical settings, we evaluated its effects on outpatient management of diabetes without remote health care professional support. In this multicenter, open-label, and single-armed prospective study, 48 patients with diabetes (including type 1 and type 2) at 3 hospitals in Japan treated with insulin or glucagon-like peptide 1 receptor agonists and performing SMBG used the app-cloud cooperation system for 24 weeks. The SMBG data were automatically uploaded to the cloud via the app. The patients could check their data, and their attending physicians reviewed the data through the cloud prior to the patients' regular visits. The primary outcome was changes in glycated hemoglobin (HbA1c) levels. Although HbA1c levels did not significantly change in all patients, the frequency of daily SMBG following applying the system was significantly increased before induction at 12 (0.60 per day, 95% CI 0.19-1.00; P=.002) and 24 weeks (0.43 per day, 95% CI 0.02-0.84; P=.04). In the subset of 21 patients whose antidiabetic medication had not been adjusted during the intervention period, a decrease in HbA1c level was observed at 12weeks (P=.02); however, this significant change disappeared at 24 weeks (P=.49). The Diabetes Treatment Satisfaction Questionnaire total score and "Q4: convenience" and "Q5: flexibility" scores significantly improved after using the system (all P<.05), and 72% (33/46) patients and 76% (35/46) physicians reported that the app-cloud cooperation system helped them adjust insulin doses. The digitization of SMBG records and sharing of the data by patients and attending physicians during face-to-face visits improved self-management in patients with diabetes. Japan Registry of Clinical Trials (jRCT) jRCTs042190057; https://jrct.niph.go.jp/en-latest-detail/jRCTs042190057.

Effect of Cytokeratin-18, C-peptide, MHR, and MACK-3 Biomarkers in Metabolic Dysfunction-Associated Fatty Liver Disease After Laparoscopic Sleeve Gastrectomy.

Laparoscopic sleeve gastrectomy (LSG) has emerged as a valuable treatment for various metabolic disorders, including metabolic dysfunction-associated fatty liver disease (MAFLD) in patients with obesity. Consequently, there is a pressing need to develop noninvasive biomarkers for diagnosing and monitoring disease progression. This study aimed to evaluate specific biomarkers, including Cytokeratin-18 (CK-18), C-peptide, monocyte to HDL cholesterol ratio (MHR), and MACK-3, in patients with obesity with MAFLD undergoing LSG. A prospective cohort study on patients with obesity before and 6 months after the LSG procedure. 70 patients with obesity with confirmed MAFLD, determined by Transient Elastography (TE), were pre- and 6 months postoperatively tested. Homeostatic Model Assessment of Insulin Resistance (HOMA-IR), lipid profile, ghrelin, leptin, peptide YY, GLP-1, and liver fibrosis scores, including AST/ALT ratio (AAR), Fibrosis-4 index (FIB-4), and BARD Score were tested. BMI significantly decreased in all participants, with a % excess weight loss of 62.0% ± 15.4%. TE measurements revealed a significant postoperative reduction from 100% to 87.1% (P = .006). All selected biomarkers showed significant postoperative improvement-a significant association of CK-18 with MAFLD markers, including AAR, FIB-4, and BARD score, were found. MACK-3 had positive associations with FIB-4. C-peptide and MHR showed no association with MAFLD markers. Furthermore, there was a positive correlation between CK-18 and MACK-3 tests and between C-peptide and CK-18 and MACK-3. Additionally, a receiver operating characteristic (ROC) curve was constructed, with CK-18 performing the best, with an estimated area under the curve of 0.863. Serum CK-18 outperformed other selected biomarkers in predicting and monitoring MAFLD in patients with obesity, suggesting its prospective utility in clinical practice. Further studies are needed to validate the accuracy of the MACK-3 test.

Insulin Secretion in Extended Mixed-Meal Tolerance Test in the Context of the Four Phases of Digestion

Background: A sequential response of insulin secretion is likely to follow the sequential pattern of digestion to optimize the effect of insulin and other counter regulatory peptides on the target tissues to maintain optimum insulin signaling pathway activation. Therefore, it is likely to relate insulin secretory response to the corresponding phase of digestion. Patients and methods: Plasma glucose, insulin and glucagon levels of three groups, each comprises of 10 persons. Group “N” for normal, group “P” for pre-diabetic and group “D” for type 2 diabetes individuals. Fluctuations in insulin secretion, in relation to changes of plasma glucose levels throughout the four phases of digestion will be traced, changes in glucagon levels will be also evaluated. Results: Early in the gastric-filling phase insulin was decreasing while glucose was rising, whereas, early in the gastric-emptying phase, insulin was rising while glucose was decreasing. Statistical significance of glucagon changes was higher than that of insulin between groups. Conclusion: Factors, which regulate insulin secretion during the gastric-filling phase, may differ than those during the gastric emptying-phase. Insulin/Glucagon ratio, is more contributed, than insulin alone, to the metabolic abnormalities associated with diabetes. It is more informative to consider insulin secretion in relation to the corresponding phase of digestion. Therefore, a four- phase cycle of insulin secretion may be suggested.

The Potential of Sheep or Camel Milk Constituents to Contribute to Novel Dressings for Diabetic Wounds.

Impaired wound healing is a complication of diabetes, which constitutes a serious problem in clinical practice. Currently, there is a high demand on the market for local treatment options for difficult-to-heal wounds caused by diabetes. The development of dressings that accelerate wound healing has recently been the subject of much research. Sheep and camel milk is gaining importance due to the content of many bioactive substances with health-promoting effects, such as insulin, LF, proline, or CLA. Sheep and camel milk proteins are a promising source of insulin, antidiabetic, and antihypertensive peptides. Numerous studies show that local administration of insulin has a significant impact on the healing of diabetic wounds. Sheep and camel milk, due to the highest LF content among ruminants, reduces autoimmune inflammatory processes and protects against bacterial and viral infections in the wound environment. Sheep's milk has the highest content of proline and CLA, and their addition to a hydrogel dressing can help in the development of an effective dressing material. The production of hydrogel dressings containing sheep and camel milk, which are naturally rich in the bioactive substances presented in this review, may be a promising step in the market of specialized dressings for difficult-to-heal diabetic wounds.

A group of pancreatic islet β cell‐like neurons are vulnerable to energy deficiency

AbstractBackgroundAlzheimer’s disease (AD) bear resemblance to the diabetes mellitus for that both of them exhibit beta‐amyloid (Aβ) deposition, insulin resistance and glycometabolic disorder. Peripheral islet β cells produce insulin and islet amyloid peptides, but whether there is a group of neurons in the brain like islet β cells is not clear.MethodHybridize APP/PS1 transgenic mice and CaMKII‐CreERT2/+ ;Tpkfl/fl strain, with tamoxifen induced Tpk knockout at 6 months of age, to form a model of energy deficiency. Labeling neuron and microglia with NeuN and Iba1 antibody, and immunofluorescence was used to observe Aβ, insulin‐like growth factor expression. TUNEL staining was applied to test cell apoptosis.ResultWe found that layer 5 cortical pyramidal neurons are strongly marked by Aβ antibody, 4G8, in APP/PS1 transgenic mice. Immunofluorescence showed that these 4G8‐positive neurons colocalized with insulin‐like growth factor 1/2 and highly expressed prohormone convertase 2. Further, we conditionally knocked out Tpk gene, a key gene involved in glucose metabolism, in excitatory neurons of APP/PS1 mice. Over time, NeuN and Iba1 labeling showed that the significant loss of neuron and activation of microglia happened in layer 5 cerebral cortex at the earliest. Additionally, the 4G8‐positive neurons in this layer were stained by TUNEL and markedly reduced in the early stage.ConclusionWe found a group of islet β cell‐like neurons that are vulnerable to glucose energy impairment, suggesting a group of vulnerable neurons in sporadic AD, and implicating the cytological mechanism of association between AD and diabetes.

Sustained hyperglycemia specifically targets translation of mRNAs for insulin secretion.

Pancreatic β cells are specialized for coupling glucose metabolism to insulin peptide production and secretion. Acute glucose exposure robustly and coordinately increases translation of proinsulin and proteins required for secretion of mature insulin peptide. By contrast, chronically elevated glucose levels that occur during diabetes impair β cell insulin secretion and have been shown experimentally to suppress insulin translation. Whether translation of other genes critical for insulin secretion is similarly downregulated by chronic high glucose is unknown. Here, we used high-throughput ribosome profiling and nascent proteomics in MIN6 insulinoma cells to elucidate the genome-wide impact of sustained high glucose on β cell mRNA translation. Before induction of ER stress or suppression of global translation, sustained high glucose suppressed glucose-stimulated insulin secretion and downregulated translation of not only insulin, but also mRNAs related to insulin secretory granule formation, exocytosis, and metabolism-coupled insulin secretion. Translation of these mRNAs was also downregulated in primary rat and human islets following ex vivo incubation with sustained high glucose and in an in vivo model of chronic mild hyperglycemia. Furthermore, translational downregulation decreased cellular abundance of these proteins. Our study uncovered a translational regulatory circuit during β cell glucose toxicity that impairs expression of proteins with critical roles in β cell function.

Neprilysin inhibitors and risk of Alzheimer's disease: A future perspective.

Alzheimer's disease (AD) is a heterogeneous neurodegenerative disease with multifaceted neuropathological disorders. AD is characterized by intracellular accumulation of phosphorylated tau proteins and extracellular deposition of amyloid beta (Aβ). Various protease enzymes, including neprilysin (NEP), are concerned with the degradation and clearance of Aβ. Indeed, a defective neuronal clearance pathway due to the dysfunction of degradation enzymes might be a possible mechanism for the accumulation of Aβ and subsequent progression of AD neuropathology. NEP is one of the most imperative metalloproteinase enzymes involved in the clearance of Aβ. This review aimed to highlight the possible role of NEP inhibitors in AD. The combination of sacubitril and valsartan which is called angiotensin receptor blocker and NEP inhibitor (ARNI) may produce beneficial and deleterious effects on AD neuropathology. NEP inhibitors might increase the risk of AD by the inhibition of Aβ clearance, and increase brain bradykinin (BK) and natriuretic peptides (NPs), which augment the pathogenesis of AD. These verdicts come from animal model studies, though they may not be applied to humans. However, clinical studies revealed promising safety findings regarding the use of ARNI. Moreover, NEP inhibition increases various neuroprotective peptides involved in inflammation, glucose homeostasis and nerve conduction. Also, NEP inhibitors may inhibit dipeptidyl peptidase 4 (DPP4) expression, ameliorating insulin and glucagon-like peptide 1 (GLP-1) levels. These findings proposed that NEP inhibitors may have a protective effect against AD development by increasing GLP-1, neuropeptide Y (NPY) and substance P, and deleterious effects by increasing brain BK. Preclinical and clinical studies are recommended in this regard.

The Insulin-Degrading Enzyme from Structure to Allosteric Modulation: New Perspectives for Drug Design.

The insulin-degrading enzyme (IDE) is a Zn2+ peptidase originally discovered as the main enzyme involved in the degradation of insulin and other amyloidogenic peptides, such as the β-amyloid (Aβ) peptide. Therefore, a role for the IDE in the cure of diabetes and Alzheimer's disease (AD) has been long envisaged. Anyway, its role in degrading amyloidogenic proteins remains not clearly defined and, more recently, novel non-proteolytic functions of the IDE have been proposed. From a structural point of view, the IDE presents an atypical clamshell structure, underscoring unique enigmatic enzymological properties. A better understanding of the structure-function relationship may contribute to solving some existing paradoxes of IDE biology and, in light of its multifunctional activity, might lead to novel therapeutic approaches.

Hybrid insulin peptide isomers spontaneously form in pancreatic beta-cells from an aspartic anhydride intermediate

Hybrid insulin peptides (HIPs) form in beta-cells when insulin fragments link to other peptides through a peptide bond. HIPs contain nongenomic amino acid sequences and have been identified as targets for autoreactive T cells in type 1 diabetes. A subgroup of HIPs, in which N-terminal amine groups of various peptides are linked to aspartic acid residues of insulin C-peptide, was detected through mass spectrometry in pancreatic islets. Here, we investigate a novel mechanism that leads to the formation of these HIPs in human and murine islets. Our research herein shows that these HIPs form spontaneously in beta-cells through a mechanism involving an aspartic anhydride intermediate. This mechanism leads to the formation of a regular HIP containing a standard peptide bond as well as a HIP-isomer containing an isopeptide bond by linkage to the carboxylic acid side chain of the aspartic acid residue. We used mass spectrometric analyses to confirm the presence of both HIP isomers in islets, thereby validating the occurrence of this novel reaction mechanism in beta-cells. The spontaneous formation of new peptide bonds within cells may lead to the development of neoepitopes that contribute to the pathogenesis of type 1 diabetes as well as other autoimmune diseases.

Decelerate amyloid fibrillation by the alkaloids extracted from Stephania venosa

Background: Naturally occurring phytochemical compounds have received considerable attention as alternative candidates for anti-amyloidogenic agents. Objectives: This study, utilizing human insulin and amyloid-β peptide as an in vitro model, determined the anti-amyloid effects of alkaloids extracted derived from Stephania venosa. Materials and methods: Alkaloids extracts including crebanine, O-methylbulbocapnine, tetrahydropalmatine and N-methyltetrahydropalmatine were used. The Inhibition of amyloid protein aggregation was studied by fluorescence spectroscopy. Results: Most alkaloids, except N-methyltetrahydropalmatine, exhibited inhibitory properties against amyloid fibrillation either insulin or amyloid-beta peptide. Among the alkaloids group, crebanine and tetrahydropalmatine showed potent properties of anti-amyloidogenesis. Conclusion: These results suggest that alkaloids could be used as a natural compound for the development of drugs against amyloid protein aggregation for the treatment of amyloid-related diseases.

Identification of Autoantibodies to a Hybrid Insulin Peptide in Type 1 Diabetes.

Type 1 diabetes (T1D) is a chronic autoimmune disease that attacks the insulin-producing b cells of the pancreatic islets. Autoantibodies to b cell proteins typically appear in the circulation years before disease onset, and serve as the most accurate biomarkers of T1D risk. Our laboratory has recently discovered novel b cell proteins comprising hybrid proinsulin:islet amyloid polypeptide peptides (IAPP). T cells from a diabetic mouse model and T1D patients are activated by these hybrid peptides. In this study, we asked whether these hybrid molecules could serve as antigens for autoantibodies in T1D and prediabetic patients. We analyzed sera from T1D patients, prediabetics and healthy age-matched donors. Using a highly sensitive electrochemiluminescence assay, sera were screened for binding to recombinant proinsulin:IAPP probes or truncated derivatives. Our results show that sera from T1D patients contain antibodies that bind larger hybrid proinsulin:IAPP probes, but not proinsulin or insulin, at significantly increased frequencies compared to normal donors. Examination of sera from prediabetic patients confirms titers of antibodies to these hybrid probes in more than 80% of individuals, often before seroconversion. These results suggest that hybrid insulin peptides are common autoantigens in T1D and prediabetic patients, and that antibodies to these peptides may serve as valuable early biomarkers of the disease.

Structural plasticity in I-Ag7 links autoreactivity to hybrid insulin peptides in type I diabetes

Structural plasticity in I-Ag7 links autoreactivity to hybrid insulin peptides in type I diabetes

Molecular characterization of four genes encoding abalone insulin-related peptides and their roles in regulation of hemolymph glucose in the Pacific abalone Haliotis discus hannai

Members of the insulin superfamily play key roles in metabolism, growth, reproduction, and longevity via insulin/IGF signaling pathway. The presence of insulin and insulin-related peptides are found among animals. In Mollusca, molluscan insulin-related peptides (MIPs) are involved in their body and shell growth. Hence, this study aimed to understand if there was difference in these peptide levels of fast and slow growing Pacific abalones, Haliotis discus hannai. To this end, four abalone insulin-related peptides (AIPs) are mined from cerebral ganglia transcriptomes of fast-growing and slow-growing abalones: AIP1, −2, −3 and −6. Notably, these AIPs possess 8 cysteine residues in their predicted insulin A and B chains, potentially supporting four disulfide bridges. Further sequence analysis based on the phylogenetic tree and multiple alignment methods indicated that AIPs have sequence features that are conserved among MIPs in many gastropods. The tissue distributions of four AIPs showed ubiquitous and similar expression in the following tissues including the cerebral ganglion, hepatopancreas, gonad, and mantle of both genders. However, there is some difference in other tissue distribution. Since the food consumption is intimately associated with the hemolymph glucose and animal growth, relative AIP1, −2, −3 and − 6 expression levels in the cerebral ganglion were measured following seaweed supplementation. The animals fed with brown seaweed showed increased these AIPs by 1.6–4.7 folds from 3 h after feeding, which lasting to 12 h, coinciding with the peak of hemolymph glucose. Based on these results, we propose that a physiological glucose regulatory mechanism involving AIPs exists in the cerebral ganglion of the H. discus hannai neuroendocrine system.

Regulatory T cells targeting a pathogenic MHC class II: Insulin peptide epitope postpone spontaneous autoimmune diabetes.

In spontaneous type 1 diabetes (T1D) non-obese diabetic (NOD) mice, the insulin B chain peptide 9-23 (B:9-23) can bind to the MHC class II molecule (IAg7) in register 3 (R3), creating a bimolecular IAg7/InsulinB:9-23 register 3 conformational epitope (InsB:R3). Previously, we showed that the InsB:R3-specific chimeric antigen receptor (CAR), constructed using an InsB:R3-monoclonal antibody, could guide CAR-expressing CD8 T cells to migrate to the islets and pancreatic lymph nodes. Regulatory T cells (Tregs) specific for an islet antigen can broadly suppress various pathogenic immune cells in the islets and effectively halt the progression of islet destruction. Therefore, we hypothesized that InsB:R3 specific Tregs would suppress autoimmune reactivity in islets and efficiently protect against T1D. To test our hypothesis, we produced InsB:R3-Tregs and tested their disease-protective effects in spontaneous T1D NOD.CD28-/- mice. InsB:R3-CAR expressing Tregs secrete IL-10 dominated cytokines upon engagement with InsB:R3 antigens. A single infusion of InsB:R3 Tregs delayed the onset of T1D in 95% of treated mice, with 35% maintaining euglycemia for two healthy lifespans, readily home to the relevant target whereas control Tregs did not. Our data demonstrate that Tregs specific for MHC class II: Insulin peptide epitope (MHCII/Insulin) protect mice against T1D more efficiently than polyclonal Tregs lacking islet antigen specificity, suggesting that the MHC II/insulin-specific Treg approach is a promising immune therapy for safely preventing T1D.

Modulation of the Activity of the Insulin-Degrading Enzyme by Aβ Peptides.

The insulin-degrading enzyme (IDE) is an evolutionarily conserved protease implicated in the degradation of insulin and amyloidogenic peptides. Most of the biochemical and biophysical characterization of IDE's catalytic activity has been conducted using solutions containing a single substrate, i.e., insulin or Aβ(1-40). IDE's activity toward a particular substrate, however, is likely to be influenced by the presence of other substrates. Here, we show by a kinetic assay based on insulin's helical circular dichroic signal and MALDI TOF mass spectrometry that Aβ peptides modulate IDE's activity toward insulin in opposing ways. Aβ(1-40) enhances IDE-dependent degradation of insulin, whereas Aβ(pyroE3-42), the most pathogenic pyroglutamate-modified Aβ peptide in AD, inhibits IDE's activity. Intriguingly, Aβ(pyroE3-42) also inhibits IDE's ability to degrade Aβ(1-40). Together, our results implicate Aβ peptides in the abnormal catabolism of IDE's key substrates.

Brush border enzyme hydrolysis and glycaemic effects of isomaltulose compared to other saccharides in dogs.

Digestible carbohydrates differ in glycaemic response, therewith having the potential to influence metabolic conditions such as insulin resistance and diabetes mellitus. Isomaltulose has been proven to lower the glycaemic response in humans, which to date has not been studied in dogs. Therefore, the aim of the present study was to characterise the digestibility, as well as the physiological effects of isomaltulose in dogs, in comparison to other saccharides. To this end, three studies were performed. Study 1 was an in vitro study, evaluating the small intestinal hydrolysis of isomaltulose compared to other relevant carbohydrate sources. Three of these saccharides, having close and low-moderate degrees of hydrolysis by brush border enzymes, were also evaluated in vivo for their glycaemic effects by measuring plasma levels of glucose, insulin and glucagon-like peptide 1 (GLP-1) 0-180 min after administration of a single dosage after an overnight fast (i.e., isomaltulose, sucrose and maltodextrin in a 3 × 3 Latin-square design, in 9 dogs, Study 2). To understand if digestive enzymes, underlying glycaemic responses for isomaltulose and sucrose can be upregulated, we exposed dogs to these saccharides for 2 weeks and repeated the measurements after an overnight fast in 18 dogs (Study 3). Isomaltulose was hydrolysed by intestinal enzyme preparation from all three dogs, but the degrading activity was low (e.g., 3.95 ± 1.03 times lower vs. sucrose), indicating a slower rate of hydrolysis. Isomaltulose had a low glycaemic response, in line with in vitro data. In vitro hydrolysis of sucrose was comparable or even higher than maltodextrin in contrast to the more pronounced glycaemic response to maltodextrin observed in vivo. The numerically higher blood glucose response to sucrose after continuous consumption, might indicate an adaptive response. In conclusion, the current work provides valuable insights into the digestion physiology of various saccharides in dogs. Further investigations on related benefits are thus warranted.

84-LB: Intensification of Glucagon-Like Peptide-1 Receptor Agonists Therapy with Glargine 300 U/mL or IDegLira—The RESTORE-G Real-World Study

Combined use of basal insulin (BI) and glucagon-like peptide 1 receptor agonist (GLP1-RA) deserves consideration for the synergistic effects on glucose profile. Nevertheless, it is unclear if the addition of BI to the ongoing GLP1-RA therapy in free or fixed combination is associated with different outcomes. Aim of this RESTORE-G sub-study was to perform a propensity-score adjusted comparative effectiveness analysis of the two most innovative BIs for intensifying GLP1-RA therapy (±oral agents), i.e. Glargine 300 U/mL in free combination (Gla-300) vs. the fixed ratio combination of insulin degludec + liraglutide (IDegLira). Table shows sample sizes, patients characteristics and effectiveness results. Compared to IDegLira, Gla-300 group had older age, worse metabolic control, and higher starting BI doses. After 6 months, statistically significant larger FBG reduction and BI dose increase were found in the free vs. fixed combination group. Trends of larger HbA1c and weight reductions with the free combination were also found, without reaching the statistical significance. In conclusion, higher benefits on FBG control in free vs. fixed combination has been found in people uncontrolled on GLP-1 RA, likely due to a better titration of the components. However, inertia in insulin initiation was documented in both groups. Disclosure R. Candido: None. A. Nicolucci: Board Member; AstraZeneca, Research Support; Novo Nordisk, Sanofi, Sobi, Shionogi &amp; Co., Ltd., AlfaSigma, Speaker's Bureau; Boehringer Ingelheim and Eli Lilly Alliance. M. Rossi: Research Support; Sanofi, Novo Nordisk, Shionogi &amp; Co., Ltd., AlfaSigma. M. Larosa: None. R. Napoli: Advisory Panel; Sanofi, Eli Lilly and Company, Novo Nordisk, Research Support; Novo Nordisk, AstraZeneca, Speaker's Bureau; Sanofi, Eli Lilly and Company, Novo Nordisk, AstraZeneca, MSD Life Science Foundation. Restore-g study group: n/a. Funding Sanofi

295-OR: Mechanisms of Antigen-Specific Immunotherapy following Tolerance Induction to a CD4 Hybrid Insulin Peptide Epitope and the Impact on CD8 T Cell Dysfunction

Type 1 diabetes is the culmination of T cell mediated autoimmunity resulting in the destruction of insulin-producing beta cells. Islet-derived hybrid insulin peptides (HIPs), recognized by autoreactive CD4 T cells, are likely dominant T cell epitopes in the NOD mouse model of autoimmune diabetes. The 2.5HIP is formed by the fusion of a fragment of insulin C-peptide with a natural cleavage product of chromogranin A, generating a neoepitope. When the 2.5HIP was delivered as an immunotherapy on tolerance inducing PLG-nanoparticles, islet grafts functioned longer in transplanted, diabetic NOD mice. Tolerance induction to this single neoepitope induced dysfunctional cytokine production in autoreactive CD4 and CD8 T cells specific for other known islet antigens. Here we used both prediabetic NOD mice and diabetic transplant recipients to examine mechanisms of peripheral tolerance induction to a HIP. Using the Tiger IL10-GFP reporter mouse, we observed a substantial increase in the proportion of (CD25+) Treg and (CD25-) Tr1 2.5HIP tetramer+ CD4 T cells that expressed IL10 in the islets, spleen, and draining lymph node of 2.5HIP nanoparticle treated mice. Autoreactive, IGRP tetramer+ CD8 T cells displayed a trafficking defect and accumulated in secondary lymphoid tissue with a concomitant reduction within islets. The proportion of cytolytic, CX3CR1+ IGRP tetramer+ CD8 T cells that infiltrated islets was also reduced. IL10 generated by both Treg and Tr1 2.5HIP tet+ T cells may directly suppress IGRP tetramer+ CD8 T cells or suppress an intermediate cell type such as a dendritic cell, causing CD8 T cell dysfunction. Examining how antigen-specific immunotherapies suppress multiple autoreactive T cells specificities may help inform future therapies without the need for immunosuppression. Disclosure J.E.Dilisio: None. R.L.Baker: None. K.M.Haskins: None. Funding National Institutes of Health (5T32DK120520-03, 2R01DK081166-11); JDRF (2-SRA-2020-907-S-B)

296-OR: Submicron Ultrasound Contrast Agents as Therapeutic Delivery Vehicles in Type 1 Diabetes

Type 1 diabetes (T1D) is characterized by immune-mediated destruction of insulin-producing beta cells resulting in severe hyperglycemia if untreated. Prior work has shown that islet microvasculature becomes hyperpermeable during presymptomatic T1D, allowing for ‘nanobubble (NB)’ submicron ultrasound contrast agents to accumulate in immune-infiltrated islets, which can be quantified using ultrasound contrast. We investigated the utility of these lipid-shelled NBs as peptide delivery vehicles to provide islet-targeted delivery, for acoustically driven therapeutic release and where therapeutic delivery can be visualized in real-time with ultrasound. We developed this platform to enhance efficacy of insulin peptide mimotopes, which can promote the expansion of anti-inflammatory regulatory T cells (Tregs) and prevent T1D in mice. We modified insulin B:9-23 peptides with a palmitoyl fatty acid tail to incorporate in the NB lipid shell. NBs accumulated specifically in NOD islets and not in other abdominal organs or NOD-SCID (control) islets (p &amp;lt; 0.0001). We found that palmitoylation enhanced peptide retention in NBs (p &amp;lt; 0.001) and is essential for delivery to NOD islets (p &amp;lt; 0.0001), where the extent of peptide delivery correlates with extent of insulitis (p &amp;lt; 0.05). Peptide incorporation did not affect NB echogenicity (in vitro and in vivo), ability to burst, and size distribution. Palmitoylation and incorporation in NBs does not affect the ability for peptide-pulsed antigen presenting cells to stimulate insulin-reactive T cells in vitro (p &amp;lt; 0.0001). Finally, NOD mice treated with peptide-NBs had a higher proportion of Tregs amongst insulin-reactive CD4+ T cells in islets, as compared to mice treated with NBs or peptide alone (p &amp;lt; 0.05). This serves as an important proof-of-principle for the usage of NBs for targeted peptide delivery and has the potential to improve therapeutic intervention in T1D. Disclosure M.Ciccaglione: None. K.Mcdaniel: None. A.W.Michels: Stock/Shareholder; Immnomolecular Therapeutics. A.A.Exner: None. R.K.Benninger: None. Funding National Institute of Diabetes and Digestive and Kidney Diseases (R01DK102950, R01DK106412, P30DK116073, T32DK120520); National Center for Advancing Translational Sciences (TL1TR002533); JDRF (1-INO-2017-435-A-N); University of Colorado

Sulfonate-Modified Polystyrene Nanoparticle at Precited Environmental Concentrations Induces Transgenerational Toxicity Associated with Increase in Germline Notch Signal of Caenorhabditis elegans.

Recently, the transgenerational toxicity of nanoplastics has received increasing attention. Caenorhabditis elegans is a useful model to assess the transgenerational toxicity of different pollutants. In nematodes, the possibility of early-life exposure to sulfonate-modified polystyrene nanoparticle (PS-S NP) causing transgenerational toxicity and its underlying mechanisms were investigated. After exposure at the L1-larval stage, transgenerational inhibition in both locomotion behavior (body bend and head thrash) and reproductive capacity (number of offspring and fertilized egg number in uterus) was induced by 1-100 μg/L PS-S NP. Meanwhile, after exposure to 1-100 μg/L PS-S NP, the expression of germline lag-2 encoding Notch ligand was increased not only at the parental generation (P0-G) but also in the offspring, and the transgenerational toxicity was inhibited by the germline RNA interference (RNAi) of lag-2. During the transgenerational toxicity formation, the parental LAG-2 activated the corresponding Notch receptor GLP-1 in the offspring, and transgenerational toxicity was also suppressed by glp-1 RNAi. GLP-1 functioned in the germline and the neurons to mediate the PS-S NP toxicity. In PS-S NP-exposed nematodes, germline GLP-1 activated the insulin peptides of INS-39, INS-3, and DAF-28, and neuronal GLP-1 inhibited the DAF-7, DBL-1, and GLB-10. Therefore, the exposure risk in inducing transgenerational toxicity through PS-S NP was suggested, and this transgenerational toxicity was mediated by the activation of germline Notch signal in organisms.