Showing papers on "Proinsulin published in 2023"

Loci for insulin processing and secretion provide insight into type 2 diabetes risk

Abstract: Insulin secretion is critical for glucose homeostasis, and increased levels of the precursor proinsulin relative to insulin indicate pancreatic islet beta-cell stress and insufficient insulin secretory capacity in the setting of insulin resistance. We conducted meta-analyses of genome-wide association results for fasting proinsulin from 16 European-ancestry studies in 45,861 individuals. We found 36 independent signals at 30 loci (p value < 5 × 10-8), which validated 12 previously reported loci for proinsulin and ten additional loci previously identified for another glycemic trait. Half of the alleles associated with higher proinsulin showed higher rather than lower effects on glucose levels, corresponding to different mechanisms. Proinsulin loci included genes that affect prohormone convertases, beta-cell dysfunction, vesicle trafficking, beta-cell transcriptional regulation, and lysosomes/autophagy processes. We colocalized 11 proinsulin signals with islet expression quantitative trait locus (eQTL) data, suggesting candidate genes, including ARSG, WIPI1, SLC7A14, and SIX3. The NKX6-3/ANK1 proinsulin signal colocalized with a T2D signal and an adipose ANK1 eQTL signal but not the islet NKX6-3 eQTL. Signals were enriched for islet enhancers, and we showed a plausible islet regulatory mechanism for the lead signal in the MADD locus. These results show how detailed genetic studies of an intermediate phenotype can elucidate mechanisms that may predispose one to disease.

Condensation of the β‐cell secretory granule luminal cargoes pro/insulin and ICA512 RESP18 homology domain

Abstract: ICA512/PTPRN is a receptor tyrosine‐like phosphatase implicated in the biogenesis and turnover of the insulin secretory granules (SGs) in pancreatic islet beta cells. Previously we found biophysical evidence that its luminal RESP18 homology domain (RESP18HD) forms a biomolecular condensate and interacts with insulin in vitro at close‐to‐neutral pH, that is, in conditions resembling those present in the early secretory pathway. Here we provide further evidence for the relevance of these findings by showing that at pH 6.8 RESP18HD interacts also with proinsulin—the physiological insulin precursor found in the early secretory pathway and the major luminal cargo of β‐cell nascent SGs. Our light scattering analyses indicate that RESP18HD and proinsulin, but also insulin, populate nanocondensates ranging in size from 15 to 300 nm and 10e2 to 10e6 molecules. Co‐condensation of RESP18HD with proinsulin/insulin transforms the initial nanocondensates into microcondensates (size >1 μm). The intrinsic tendency of proinsulin to self‐condensate implies that, in the ER, a chaperoning mechanism must arrest its spontaneous intermolecular condensation to allow for proper intramolecular folding. These data further suggest that proinsulin is an early driver of insulin SG biogenesis, in a process in which its co‐condensation with RESP18HD participates in their phase separation from other secretory proteins in transit through the same compartments but destined to other routes. Through the cytosolic tail of ICA512, proinsulin co‐condensation with RESP18HD may further orchestrate the recruitment of cytosolic factors involved in membrane budding and fission of transport vesicles and nascent SGs.

Tirzepatide as Monotherapy Improved Markers of Beta-cell Function and Insulin Sensitivity in Type 2 Diabetes (SURPASS-1)

Abstract: Abstract Context Tirzepatide is a glucose-dependent insulinotropic polypeptide and glucagon-like peptide-1 receptor agonist approved for treatment of type 2 diabetes (T2D). SURPASS-1, a phase 3 trial of tirzepatide monotherapy in people with early T2D, enables evaluating effects of tirzepatide on pancreatic beta-cell function and insulin sensitivity (IS) without other background antihyperglycemic medications. Objective Explore changes in biomarkers of beta-cell function and IS with tirzepatide monotherapy. Design Post hoc analyses of fasting biomarkers with analysis of variance and mixed model repeated measures. Setting Forty-seven sites in 4 countries. Patients Four hundred seventy-eight T2D participants. Intervention Tirzepatide (5, 10, 15 mg), placebo. Main Outcome Measure(s) Analyze biomarkers of beta-cell function and IS at 40 weeks. Results At 40 weeks, markers of beta-cell function improved with tirzepatide monotherapy vs placebo with reductions from baseline in fasting proinsulin levels (49-55% vs −0.6%) and in intact proinsulin/C-peptide ratios (47-49% vs −0.1%) (P < .001, all doses vs placebo). Increases from baseline in homeostatic model assessment for beta-cell function (computed with C-peptide) (77-92% vs −1.4%) and decreases in glucose-adjusted glucagon levels (37-44% vs +4.8%) were observed with tirzepatide vs placebo (P < .001, all doses vs placebo). IS improved as indicated by reductions from baseline in homeostatic model assessment for insulin resistance (9-23% vs +14.7%) and fasting insulin levels (2-12% vs +15%), and increases in total adiponectin (16-23% vs −0.2%) and insulin-like growth factor binding protein 2 (38-70% vs +4.1%) with tirzepatide vs placebo at 40 weeks (P ≤ .031, all doses vs placebo, except for fasting insulin levels with tirzepatide 10 mg). Conclusions As monotherapy for early T2D, tirzepatide achieved significant improvements in biomarkers of both pancreatic beta-cell function and IS.

&lt;strong&gt;Deletion of carboxypeptidase E in beta cells disrupts proinsulin processing but does not lead to spontaneous development of diabetes in mice&lt;/strong&gt;

Abstract: <p>Carboxypeptidase E (CPE) facilitates the conversion of prohormones into mature hormones and is highly expressed in multiple neuroendocrine tissues. Carriers of <em>CPE</em> mutations have elevated plasma proinsulin and develop severe obesity and hyperglycemia. We aimed to determine whether loss of <em>Cpe</em> in pancreatic beta cells disrupts proinsulin processing and accelerates development of diabetes and obesity in mice. Pancreatic beta cell-specific Cpe knockout mice (β<em>Cpe</em>KO; <em>Cpe</em>fl/fl x <em>Ins1</em>Cre/+) lack mature insulin granules and have elevated proinsulin in plasma; however, glucose-and KCl-stimulated insulin secretion in β<em>Cpe</em>KO islets remained intact. High fat diet-fed β<em>Cpe</em>KO mice showed comparable weight gain and glucose tolerance compared to <em>Wt</em> littermates. Notably, beta-cell area was increased in chow-fed β<em>Cpe</em>KO mice and beta-cell replication was elevated in β<em>Cpe</em>KO islets. Transcriptomic analysis of β<em>Cpe</em>KO beta cells revealed elevated glycolysis and <em>Hif1α</em>-target gene expression. Upon high glucose challenge, beta cells from β<em>Cpe</em>KO mice showed reduced mitochondrial membrane potential, increased reactive oxygen species, reduced <em>MafA,</em> and elevated <em>Aldh1a3 </em>transcript levels. Following multiple low-dose streptozotocin treatment, β<em>Cpe</em>KO mice had accelerated hyperglycemia with reduced beta-cell insulin and Glut2 expression. These findings suggest that <em>Cpe</em> and proper proinsulin processing are critical in maintaining beta cell function during the development of hyperglycemia.</p>

Simultaneous attenuation of hyperglycemic memory-induced retinal, pulmonary, and glomerular dysfunctions by proinsulin C-peptide in diabetes

Abstract: Abstract Background Hyperglycemic memory (HGM) is a pivotal phenomenon in the development of diabetic complications. Although coincident diabetic complications are reported, research on their development and treatment is limited. Thus, we investigated whether C-peptide can simultaneously inhibit HGM-induced retinal, pulmonary, and glomerular dysfunctions in diabetic mice supplemented with insulin. Methods Insulin-treated diabetic mice were supplemented with human C-peptide by subcutaneous implantation of K9-C-peptide depots for 4 weeks, and reactive oxygen species (ROS) generation, transglutaminase (TGase) activity, and vascular leakage were examined in the retina, lung, and kidney. Results We found hyperglycemia-induced persistent ROS generation and TGase activation after blood glucose normalization in the retina, lung, and kidney of insulin-supplemented diabetic mice. These pathological events were inhibited by systemic supplementation of human C-peptide via subcutaneous implantation of a thermosensitive biopolymer-conjugated C-peptide depot. ROS generation and TGase activation were in a vicious cycle after glucose normalization, and C-peptide suppressed the vicious cycle and subsequent endothelial permeability in human retinal endothelial cells. Moreover, C-peptide supplementation ameliorated HGM-induced retinal vascular leakage and neurodegeneration, pulmonary vascular leakage and fibrosis, and glomerular adherens junction disruption and vascular leakage. Conclusions Overall, our findings demonstrate that C-peptide supplementation simultaneously attenuates vascular and neuronal dysfunctions in the retina, lung, and glomerulus of insulin-supplemented diabetic mice.

Investigating the genetic relationship between migraine and headache with glucose-related traits

Abstract: Epidemiological studies have found conflicting evidence for an association of migraine and headache with glucose-related traits, and the pathophysiological mechanisms underlying such an association remain unknown. In this PhD thesis, I showed that migraine, headache, and glycemic traits share a common genetic etiology, and identified novel genes and biological pathways jointly influencing migraine and glucose-related traits that offer novel insights into their underlying genetic mechanisms. Additionally, I found a causal relationship between headache and fasting proinsulin, suggesting that fasting proinsulin decreases the risk of headache and may offer new avenues for developing more effective preventive measures.

Optimisation of induction conditions for a bacterial strain producing proinsulin aspart

Abstract: Diabetes poses a serious threat to the health of people around the world. Therefore, in 2021, the World Health Organisation launched the Global Diabetes Compact, an initiative aimed at improving the management and prevention of diabetes. The rapid growth in the number of diabetic patients has increased the need for insulin. Rapid-acting human insulin analogues, including insulin aspart, improve the efficacy of insulin therapy. Methods for insulin aspart production include its biosynthesis in the proinsulin form in Escherichia coli. However, the yield of the recombinant protein largely depends on the optimisation of the production process.The aim of the study was to optimise the induction conditions for an E. coli strain expressing recombinant proinsulin aspart through applying the Design of Experiment (DoE) approach to enhance bacterial cell productivity.Materials and methods. The study focused on a strain of E. coli producing proinsulin aspart. The authors planned the experiment using MODDE software and the reduced face-centred central composite design (CCF) enabling the assessment of factor interactions and the creation of design spaces. The authors carried out fermentations of the producing strain in a 5 L Biostat® B bioreactor and measured proinsulin aspart concentrations by capillary gel electrophoresis. The results were analysed using GraphPad Prism 6.Results. Using the DoE approach, the authors optimised the conditions for the growth of the producer strain and the biosynthesis of proinsulin aspart. Based on data from response surface plots for wet biomass concentration, specific productivity, and volumetric productivity, as well as plotted models, the authors established design spaces for the induction of proinsulin aspart expression in E. coli. The plotted models demonstrated high predictive power and high reproducibility of the results. The authors successfully validated the induction process for the synthesis of proinsulin aspart in a bioreactor under optimised conditions. The volumetric productivity of the strain producing proinsulin aspart increased from 3.06±0.16 g/L (conventional conditions) to 4.93±0.80 g/L (optimised conditions).Conclusions. The authors achieved a 60% increase in the volumetric yield of proinsulin aspart. The study results may be used to intensify the industrial production of insulin aspart.

743-P: Delayed Diabetes with Proinsulin mRNA Vaccines in NOD Mice

Abstract: Objective: In type 1 diabetes, an immunomodulatory, antigen-specific therapy may abrogate autoimmunity and support immune tolerance to prevent β-cell destruction and disease progression. This study aims to assess the efficacy of a novel proinsulin II mRNA multi-lamellar liposome vaccine in preventing diabetes in the non-obese diabetic (NOD) mouse. We hypothesized that proinsulin mRNA vaccines would restore immunotolerance and delay diabetes. Methods: Proinsulin II was cloned into plasmids for in vitro RNA transcription. mRNA was loaded into DOTAP liposomes. mRNA expression was validated in HEK293 cell line transfection. NOD/ShiLtJ strain mice were obtained at 4 weeks of age. Group 1 received three IV tail vein injections of mRNA vaccines every other day at 8 weeks and one dose at 23 weeks of age. Group 2 remained untreated. Mice were checked weekly for diabetes (glucose ≥250 mg/dl on two measurements one day apart). Kaplan-Meier survival curves, hazard ratio, median time to diabetes, and t-tests were used to compare time to diabetes. Results: The diabetes rate by 32 weeks was 64.2% (9/14) in group 1 compared to 78.6% (11/14) in group 2 (Hazard ratio 1.60 95% CI 0.66-3.86). By 15 weeks, the treatment group had no diabetes compared to 35.7% untreated (p=0.04). Median time to diabetes was 23 weeks in the treatment group and 21 weeks in untreated. Conclusions: Proinsulin II mRNA multi-lamellar liposome vaccines temporarily delay diabetes in NOD mice. T.P.Foster: None. M.J.Haller: Board Member; SAB Biotherapeutics, Inc., Consultant; Sanofi, MannKind Corporation. D.Schatz: Advisory Panel; Medtronic, Avotres Inc., QuLab Medical Ltd., Kriya Therapeutics. C.Wasserfall: None. E.Sayour: None. Endocrine Fellows Foundation

Human growth hormone and insulin, from tissue extracts to recombinant DNA technology, their biosimilars and analogues in treatment of Endocrine Disorders

Abstract: Forty years ago, recombinant DNA technology caused a revolution in the way growth disorders and diabetes could be treated. Bacteria or human cells were modified so as to be able to produce human hormones instead of relying on cadaveric material in the case of growth disorders and porcine insulin in the case of diabetics. The new technology also removed the risk of contracted hepatitis or fatal Creutzfeldt Jacob disease. A signal peptide was used to transport the new protein into the periplasmic space giving rise to an exact, correctly folded version of monomeric human growth hormone. More eligible patients could be treated, resulting not only in a large number of clinical trials. Daily tiny subcutaneous injections were found to be much more effective than twice or thrice weekly in the treatment of children and adults, The progress for recombinant human insulin turned out to be somewhat different. Insulin is a more complicated protein, with an Alpha and Beta chain joined by two disulphide bridges, but is produced in the human pancreas as a single chain, proinsulin, the C peptide is removed to make the active molecule. Recombinant insulin development was in a way the opposite to growth hormone since human insulin acts faster than porcine. Short fast and long-acting analogues have been developed all to improve the treatment regimens for both type 1 and type 2 diabetics, A similar development is now taking place in the growth hormone field. Depot preparations for weekly administration are under development to achieve better compliance. One recently approved depot uses an analogue, modified in one amino acid of the peptide chain to permit acylation and albumin binding. In a way this is a retrograde step and it remains to be seen if the patient’s growth and metabolic responses are comparable to the daily administration of tiny amount of recombinant natural sequence hormone over several years of treatment.

Cross-trait analyses identify shared genetics between migraine, headache, and glycemic traits, and a causal relationship with fasting proinsulin.

Abstract: Abstract The co-occurrence of migraine and glycemic traits has long been reported in observational epidemiological studies, but it has remained unknown how they are linked genetically. We used large-scale GWAS summary statistics on migraine, headache, and nine glycemic traits in European populations to perform cross-trait analyses to estimate genetic correlation, identify shared genomic regions, loci, genes, and pathways, and test for causal relationships. Out of the nine glycemic traits, significant genetic correlation was observed for fasting insulin (FI) and glycated haemoglobin (HbA1c) with both migraine and headache, while 2-h glucose was genetically correlated only with migraine. Among 1703 linkage disequilibrium (LD) independent regions of the genome, we found pleiotropic regions between migraine and FI, fasting glucose (FG), and HbA1c, and pleiotropic regions between headache and glucose, FI, HbA1c, and fasting proinsulin. Cross-trait GWAS meta-analysis with glycemic traits, identified six novel genome-wide significant lead SNPs with migraine, and six novel lead SNPs with headache ( P meta < 5.0 × 10 –8 and P single-trait < 1 × 10 –4 ), all of which were LD-independent. Genes with a nominal gene-based association ( P gene ≤ 0.05) were significantly enriched (overlapping) across the migraine, headache, and glycemic traits. Mendelian randomisation analyses produced intriguing, but inconsistent, evidence for a causal relationship between migraine and headache with multiple glycemic traits; and consistent evidence suggesting increased fasting proinsulin levels may causally decrease the risk of headache. Our findings indicate that migraine, headache, and glycemic traits share a common genetic etiology and provide genetic insights into the molecular mechanisms contributing to their comorbid relationship.

1751-P: SERCA2 Regulates ER Lipid Composition and Proinsulin Processing

Abstract: Endoplasmic reticulum calcium (ER Ca2+) levels are primarily maintained through the sarco-ER Ca2+ ATPase (SERCA) pump. We have demonstrated reduced SERCA2 expression in islets from human cadaveric donors with Type 2 Diabetes (T2D) compared to normoglycemic controls and linked β-cell specific deletion of SERCA2 with impaired proinsulin processing. To test mechanisms of how reduced ER Ca2+ and SERCA2 deficiency regulate proinsulin processing, a SNAP-tagged human proinsulin adenovirus (SNAP-proinsulin) was overexpressed in rat insulinoma β-cells where SERCA2 had been deleted (S2KO INS-1) using CRISPR/Cas9 and in islets from C57BL/6 mice with SERCA2 haploinsufficiency fed a high fat diet or treated ex vivo with glucolipotoxicity (GLT). Immunoblotting SNAP-tagged cleavage products showed reduced proinsulin processing in SERCA2 deficient models compared to wildtype (WT), these defects were exacerbated by GLT treatment. To determine whether reduced processing could be related to impaired trafficking of proinsulin within the β-cell secretory pathway, SNAP-proinsulin was fluorescently labelled in C57BL/6 mouse islets treated with GLT. The colocalization of SNAP-proinsulin with Golgi immuno-marker, Giantin, suggested an accumulation in this compartment. The ER is the main site of lipid synthesis and lipid remodeling is known to regulate β-cell function. Preliminary mass spectrometry data revealed that loss of SERCA2 increases ER levels of phosphatidylcholine and decreases ceramide and sphingomyelin in S2KO INS-1 cells compared to WT cells. Taken together, these results demonstrate that ER Ca2+ deficiency changes ER lipid composition, which may affect proinsulin processing and trafficking. Characterizing this novel mechanism could provide valuable insight into β-cell dysfunction in T2D. M.Hartley: Employee; Eli Lilly and Company. M.J.Pearson: None. F.Syed: None. H.Bui: None. T.Kono: None. K.D.Roth: Employee; Eli Lilly and Company. C.Evans-molina: Advisory Panel; Provention Bio, Inc., DiogenX, Avotres Inc., Neurodon, MaiCell Therapeutics, Other Relationship; Isla Technology, Bristol-Myers Squibb Company, Nimbus Therapeutics, Research Support; Lilly, Astellas Pharma Inc. National Institute of Diabetes and Digestive and Kidney Diseases (R01DK093954, R01DK127308, R01DK127236)

1752-P: The Role of Translocon-Associated Protein Subunit γ in Insulin Biosynthesis and Glucose Homeostasis

Abstract: Translocation-associated protein (TRAP) is a four-subunit complex located on the endoplasmic reticulum (ER) membrane, including TRAPα, TRAPβ, TRAPγ, and TRAPδ. TRAPα, TRAPβ and TRAPδ reside on the luminal side of the ER membrane for primary functionality, whereas the extra-membranous portion of TRAPγ is mainly cytosolic. Our recent studies indicate that TRAP complex contributes to the biosynthesis of proinsulin in pancreatic β cell lines and TRAPγ is a rate-limiting element of proinsulin biosynthesis. This might be partially explained by that newly made preproinsulin may first encounter the cytosolic domain of TRAPγ for translocation into the ER. To further understand the role of TRAPγ in β cells and its pathological significance in maintaining glucose homeostasis, we generated a β-cell specific TRAPγ knockout mouse model. We found that TRAPγ deficiency led to a significant reduction of insulin content in islets and a decrease of circulating insulin. TRAPγ βKO mice impaired glucose tolerance with aging, which even further deteriorated with high fat diet. In β cell line, TRAPγ deficiency resulted in less proinsulin synthesis and poor response to high glucose stimulation. Consistently, an acute exposure to high glucose increased expression of TRAPγ and proinsulin protein levels simultaneously in wild-type mouse islets. However, the ability to upregulate proinsulin response to high glucose was markedly compromised in TRAPγ βKO mouse islets. This study uncovers that TRAPγ is critical to insulin synthesis both in vivo and in vitro, which provides insight into the pathogenesis of type 2 Diabetes. N.Xu: None. X.Xu: None. J.Hu: None. J.Guo: None. J.Cui: None. M.Liu: None. W.Feng: None. X.Li: None. National Natural Science Foundation of China (81830025, 82220108014, 82100865, 81800733, 81900720, 82000796, 82070854); National Key R&D Program of China (2019YFA0802502, 2022YFE0131400); Tianjin Key Medical Discipline Construction Project (TJYXZDXK-030A)

Ageing and Insulin: Inter Alia Diabetes, Obesity and Fitness Concerns

Abstract: This article argues the essence of insulin sensitivity versus secretion including insulin resistance in the clinical strategy for the treatment, lifestyle changes, prompt interventions and control of diabetes. Progressive decline of glucose tolerance as advanced age occurs has been associated with the pathogenesis of type 2 diabetes due to peripheral insulin resistance and impaired β cell function. In elderly persons, insulin secretion is deranged concomitantly with decreased insulin clearance rate and augmented circulating proinsulin level that ostensibly explicates age-related hyperglycemia. Insulin is associated with numerous pathophysiological processes exhibited during brain function in learning and memory, as well as the regulation of ageing, metabolic syndrome, obesity, diabetes and cardiovascular diseases in Man. Elevated chronic peripheral insulin, decreased insulin action and brain insulin contents are pathognomonic of the insulin resistance syndrome. All these are associated through specific mechanisms in the pathophysiology of ageing and insulin in concert with risk factors and the concomitant complications. Ostensibly, progressive excess insulin induces synchronous elevated levels of oxidative stress and inflammatory impacts which exacerbate or are exacerbated by advancing age, culminating as inimical consequences to healthy lifestyles, longevity or extended lifespan. Therapeutics and other healthcare measures may be beneficial in order to prevent, mitigate or amend insulin aberrations in the elderly and during the ageing process. The mainstay in managing an elderly patient with perturbed insulin action, is control of therapeutic application, as it can reverse acute terminal states. Treatment necessitates stringent and thorough expertise, knowledge and skills for optimum provision and effective cerebral, cardiovascular and skeletal protection for a healthier lifespan and longevity. This article will be of immense contribution in the understanding of the prevention, control and treatment of insulin resistance, metabolic syndrome, diabetes, obesity, cardiovascular and neurological disorders during ageing.

&lt;strong&gt;Deletion of carboxypeptidase E in beta cells disrupts proinsulin processing but does not lead to spontaneous development of diabetes in mice&lt;/strong&gt;

Abstract: <p>Carboxypeptidase E (CPE) facilitates the conversion of prohormones into mature hormones and is highly expressed in multiple neuroendocrine tissues. Carriers of <em>CPE</em> mutations have elevated plasma proinsulin and develop severe obesity and hyperglycemia. We aimed to determine whether loss of <em>Cpe</em> in pancreatic beta cells disrupts proinsulin processing and accelerates development of diabetes and obesity in mice. Pancreatic beta cell-specific Cpe knockout mice (β<em>Cpe</em>KO; <em>Cpe</em>fl/fl x <em>Ins1</em>Cre/+) lack mature insulin granules and have elevated proinsulin in plasma; however, glucose-and KCl-stimulated insulin secretion in β<em>Cpe</em>KO islets remained intact. High fat diet-fed β<em>Cpe</em>KO mice showed comparable weight gain and glucose tolerance compared to <em>Wt</em> littermates. Notably, beta-cell area was increased in chow-fed β<em>Cpe</em>KO mice and beta-cell replication was elevated in β<em>Cpe</em>KO islets. Transcriptomic analysis of β<em>Cpe</em>KO beta cells revealed elevated glycolysis and <em>Hif1α</em>-target gene expression. Upon high glucose challenge, beta cells from β<em>Cpe</em>KO mice showed reduced mitochondrial membrane potential, increased reactive oxygen species, reduced <em>MafA,</em> and elevated <em>Aldh1a3 </em>transcript levels. Following multiple low-dose streptozotocin treatment, β<em>Cpe</em>KO mice had accelerated hyperglycemia with reduced beta-cell insulin and Glut2 expression. These findings suggest that <em>Cpe</em> and proper proinsulin processing are critical in maintaining beta cell function during the development of hyperglycemia.</p>

Proinsulin-to-C-peptide ratio as a marker of beta-cell function in African American and European American adults

Abstract: <p> </p> <p><strong>Objective</strong>: The primary purpose of the present study was to test the hypothesis that the proinsulin-to-C-peptide ratio (PI:CP), as an index of proinsulin secretion, would be higher and associated with indices of beta-cell function in African American adults relative to European American adults without type 2 diabetes. </p> <p><strong>Research Design and Methods</strong>: Participants were 114 African American and European American adult men and women. A 2-h OGTT was conducted to measure glucose, insulin, C-peptide, and proinsulin, and derive indices of beta-cell response to glucose. The Matsuda index was calculated as a measure of insulin sensitivity. The disposition index (DI, the product of insulin sensitivity and beta-cell response) was calculated for each phase of beta-cell responsivity. Pearson correlations were used to investigate the relationship of PI:CP with each phase of beta-cell response (Φb, Φd, Φs, Φtot), disposition indices (DId, DIs, DItot), and insulin sensitivity. Multiple linear regression analysis was used to evaluate independent contributions of race, BMI, and glucose tolerance status on PI:CP levels, before and after adjustment for insulin sensitivity. </p> <p><strong>Results: </strong>African American participants had higher fasting and 2-h PI:CP. Fasting PI:CP was positively associated with Φb, and fasting PI:CP and 2-h PI:CP were inversely associated with DId and insulin sensitivity only in African American participants. </p> <p><strong>Conclusions: </strong>PI:CP could be useful in identifying African American individuals at highest risk for beta-cell dysfunction and ultimately type 2 diabetes. </p>

A Review of the Biosynthesis and Structural Implications of Insulin Gene Mutations Linked to Human Disease

Abstract: The discovery of the insulin hormone over 100 years ago, and its subsequent therapeutic application, marked a key landmark in the history of medicine and medical research. The many roles insulin plays in cell metabolism and growth have been revealed by extensive investigations into the structure and function of insulin, the insulin tyrosine kinase receptor (IR), as well as the signalling cascades, which occur upon insulin binding to the IR. In this review, the insulin gene mutations identified as causing disease and the structural implications of these mutations will be discussed. Over 100 studies were evaluated by one reviewing author, and over 70 insulin gene mutations were identified. Mutations may impair insulin gene transcription and translation, preproinsulin trafficking and proinsulin sorting, or insulin-IR interactions. A better understanding of insulin gene mutations and the resultant pathophysiology can give essential insight into the molecular mechanisms underlying impaired insulin biosynthesis and insulin-IR interaction.

1345-P: Proinsulin–to–C-Peptide Ratio Is Associated with the Time above the Target Glucose Range and the Duration of Type 2 Diabetes

Abstract: Background: The proinsulin-to-C-peptide (PI:C) ratio is thought to be an index of pancreatic β-cell dysfunction, and in subjects with prediabetes and type 2 diabetes (T2DM), abnormally high PI:C ratios have been identified. In the present study, we investigated the relationships of the duration of diabetes and glucose variability in subjects with T2DM with the PI:C ratio. Methods: We performed a prospective observational study of 272 Japanese outpatients with T2DM who underwent blinded professional continuous glucose monitoring (CGM) and fasting blood sample collection. Their fasting PI:C ratio and CGM-related indices were calculated. After placing all the participants into four groups on the basis of quartile (Q) of diabetes duration (Q1 <8 years, Q2 8-13 years, Q3 14-21 years, Q4 ≥22 years), the characteristics associated with PI:C ratio were identified. All of the statistical tests were two-sided and P<0.05 was considered to indicate statistical significance. Results: The mean age of the cohort was 68.0 years, their mean HbA1c 7.1%, and their mean body mass index (BMI) 24.9 kg/m2. High PI:C ratio was associated with a short duration of diabetes (P<0.001). In each group, there was a positive correlation between PI:C ratio and the time above the target glucose range (TAR; >180 mg/dL) in Q1, Q2, and Q3. The closest correlation between PI:C ratio and TAR was found for Q1 but there was no significant correlation for Q4 (Q1: ρ=0.38, P=0.003; Q2: ρ=0.28, P=0.019; Q3: ρ=0.24, P=0.045; Q4: ρ=0.03, P=0.811). Conclusions: We have shown that the PI:C ratio was positively associated with TAR in subjects with a short duration of T2DM. Given that insufficient pancreatic β-cell function and hyperglycemia is closely related, the PI:C ratio may play a role as the index of pancreatic β-cell dysfunction in subjects with a short duration of T2DM, but not in those with long-standing T2DM. A.Miya: None. A.Nakamura: Research Support; Abbott Japan Co., Ltd., Boehringer Ingelheim Japan, Inc., Daiichi Sankyo, Taisho Pharmaceutical Holdings Co., Ltd., Teijin Pharma Limited, Kowa Company, Ltd., Mitsubishi Tanabe Pharma Corporation. H.Nomoto: Speaker's Bureau; Novo Nordisk, Sumitomo Pharma, Co., Ltd. H.Kameda: None. T.Atsumi: Speaker's Bureau; Eli Lilly Japan K.K., Kowa Company, Ltd. Japan Society for the Promotion of Science (22K17766)

Nesidioblastoma (Congenital Hyperinsulinism - CHI)

Abstract: Nesidioblastoma or persistent hyper insulinemic hypoglycemia of infancy (PHHI) or congenital hyperinsulinism (CHI) is a very uncommon yet life-threatening neuroendocrine cause of hypoglycemia. Its characteristic feature is recurrent episodes of severe, sustained, or persistent hypoglycemia due to elevated insulin levels, along with elevated C-peptide and proinsulin, beyond few hours of life. If not recognized promptly and managed, it may result in long-term neurological damage in the form of seizures, mental and psychological retardation, neurological deficits and even death from undiagnosed severe hypoglycemia. It is heterogenous clinically, genetically, and histopathologically.

Proinsulin-to-C-Peptide Ratio as a Marker of β-Cell Function in African American and European American Adults.

Abstract: OBJECTIVE The primary purpose of the current study was to test the hypothesis that the proinsulin-to-C-peptide (PI-to-CP) ratio, as an index of proinsulin secretion, would be higher and associated with indices of β-cell function in African American adults relative to European American adults without type 2 diabetes. RESEARCH DESIGN AND METHODS Participants were 114 African American and European American adult men and women. A 2-h oral glucose tolerance test was conducted to measure glucose, insulin, C-peptide, and proinsulin and derive indices of β-cell response to glucose. The Matsuda index was calculated as a measure of insulin sensitivity. The disposition index (DI), the product of insulin sensitivity and β-cell response, was calculated for each phase of β-cell responsivity. Pearson correlations were used to investigate the relationship of the PI-to-CP ratio with each phase of [beta symbol]-cell response (basal, Φb; dynamic, Φd; static, Φs; total, Φtot), disposition indices (DId, DIs, DItot), and insulin sensitivity. Multiple linear regression analysis was used to evaluate independent contributions of race, BMI, and glucose tolerance status on PI-to-CP levels before and after adjustment for insulin sensitivity. RESULTS African American participants had higher fasting and 2-h PI-to-CP ratios. The fasting PI-to-CP ratio was positively associated with Φb, and the fasting PI-to-CP ratio and 2-h PI-to-CP ratio were inversely associated with DId and insulin sensitivity only in African American participants. CONCLUSIONS The PI-to-CP ratio could be useful in identifying African American individuals at highest risk for β-cell dysfunction and ultimately type 2 diabetes.

Is β-Cell Dysfunction Present in Adult Autoantibody Negative Relatives of Individuals with Type 1 Diabetes Mellitus?

Abstract: Background/Objective: Individuals with a family history of type 1 diabetes mellitus (T1D) are at increased genetic risk for T1D. Previous studies identified the presence of β-cell dysfunction before clinical onset and diagnosis of T1D. However, it is unclear if β-cell dysfunction predates islet autoimmunity in individuals at high genetic risk. Our objective was to test β-cell function in islet antibody negative adults who have a first-degree relative with T1D. We hypothesized that individuals at genetic risk for T1D would exhibit β-cell dysfunction even without detectable islet autoimmunity.Methods: We used ordinary one-way and Brown-Forsythe ANOVA to compare the repeated mixed meal tolerance test (MMTT) and hyperglycemic clamp glucose-stimulated β-cell response and function measures between three groups of individuals: normoglycemic adults without T1D family history age, sex, and BMI-matched islet antibody negative first-degree relatives of individuals with T1D, and islet antibody positive first-degree relatives of individuals with T1D.Results: Neither the MMTT first-phase insulin secretion measures (c-peptide0-15 minutes, c-peptide0-30 minutes, insulin0-15 minutes, insulin0-30 minutes), nor second-phase measures (c-peptide0-120 minutes, insulin0-120 minutes, and glucose0-120 minutes) showed a statistically significant difference between groups. The clamp acute c-peptide response to glucose, insulin sensitivity, c-peptide steady state, first-phase β-cell function, and second-phase β-cell function were similar between subject groups in both visits. Fasting proinsulin:c-peptide ratios, a biomarker of β-cell stress, were also similar between participant groups.Conclusion and Impact: Our data suggest that genetically at-risk autoantibody negative adult relatives of individuals with T1D do not demonstrate β-cell dysfunction compared to controls. Studies show that β-cell ER dysfunction preceding T1D onset is more striking in younger children. Thus, our findings may reflect the use of an adult study population. Alternatively, β-cell dysfunction in T1D may require initial autoimmune activation. This study will contribute to the growing understanding of risk factors contributing to T1D development.This project was funded, in part, with support from the Research Training Program in Diabetes and Obesity funded, in part by grant 3T32DK064466 from the National Institutes of Health. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Funding also provided by JDRF grant 2-SRA-2017-498-M-B.

1657-P: Post–Mixed Meal Glucagon Secretion Is Not Influenced by Residual Beta-Cell Function in Type 1 Diabetes

Abstract: Aims: Postprandial glucose excursions contribute to dysglycemia in type 1 diabetes (T1D). Hyperglucagonaemia may play a role but the relationship between beta and alpha-cell dysfunction together with incretin response remains unclear. We sought to elucidate this in people with established T1D with and without C-peptide. Methods: Participants (n=29, age 38 ± 12 years, HbA1c 57 ± 9 mmol/mol, T1D duration 20 ± 13 years) with a range of postprandial urine C-peptide were recruited. A Meal Tolerance Test (MTT, 240 mL Fortisip: 360kcal, 14.4g protein, 13.92g fat, 44.16g carbohydrate) was completed after an overnight fast, maintaining normal basal insulin, without an insulin bolus. Blood samples were taken pre and 30min intervals for 180 minutes post-drink and analysed for glucose, C-peptide, glucagon, proinsulin, GLP-1 and GIP. Participants were grouped by peak C-peptide (high >200 pmol/L, low 3-200pmol/L, undetectable). [Incremental] area under the curves ([i]AUC) were compared by one way ANOVA, Spearman's correlation and multiple regression with significance accepted p≤0.05. Results: Ten participants had high peak C-peptide (680 ± 426 pmol/L), 8 low (39 ± 34 pmol/L), and 11 undetectable. Glucagon was comparable in all groups with mean AUC 1721 ± 890 pmol/L*180min (p=0.84). Glucagon correlated with GLP-1 (r=0.81, p<0.01) and GIP (r=0.43, p=0.02) response, but not C-peptide (R=-0.10, p=0.61), proinsulin (r=-0.18, p=0.35) or proinsulin/C-peptide ratio (r=0.04, p=0.85). C-peptide predicted glucose AUC using multiple regression (R2 = 44.3%, F(1, 27) = 21.4, p<0.01). C-peptide in combination with glucagon predicted glucose iAUC (R2 = 41.1% F(2, 26) = 9.1, p<0.01). Conclusion: Residual C-peptide is a predictor of MTT glucose excursions but not glucagon response in established T1D. Postprandial glucagon contributed to glucose excursion as well as correlating with incretin response. This supports altered alpha-cell phenotype in T1D, aberrantly responding to or secreting incretin. G.Taylor: None. K.Smith: None. A.Bashir: None. T.J.Mcdonald: None. E.J.Stevenson: None. J.A.Shaw: Advisory Panel; Betalin Therapeutics, Provention Bio, Inc., Consultant; Mogrify. D.J.West: None. Diabetes Research and Wellness Foundation (SCA/OF/12/15); Francis James Bell Endowment Fund; Country Durham Community Foundation

236-OR: Pancreatic ß-Cell TRAPδ Deficiency Reduces Insulin Production but Improves Insulin Sensitivity

Abstract: The endoplasmic reticulum (ER) membrane protein translocon-associated protein-δ (TRAPδ, also known as SSR4) is involved in insulin biosynthesis in pancreatic β cell lines. However, its pathophysiological significance for the maintenance of islet β cell function and glucose homeostasis is unclear. Herein, we generated a mouse line with pancreatic β cell-specific deletion of TRAPδ (TRAPδ βKO), and found that TRAPδ βKO led to decreased circulating insulin in mice fed with either normal chow diet or high fat diet. Multiple independent experiments confirmed that, although TRAPδ deletion decreased insulin content in the islets, it did not affect composition of islet cells, insulin gene expression, insulin/proinsulin ratio, nor the expression and glycosylation of the prohormone enzymes involved in proinsulin processing. These data suggest that TRAPδ does not play a critical role in the transcription of insulin gene and processing of proinsulin. Importantly, untranslocated preproinsulin was significantly increased when the islets were treated with a proteasomal inhibitor, suggesting that TRAPδ deficiency impaired preproinsulin translocation, resulting in a rapid degradation of untranslocated preproinsulin that accounts for the decreased insulin production. Interestingly, the moderate decrease in the circulating insulin level in TRAPδ βKO mice did not cause impaired glucose tolerance and elevated fasting blood glucose, suggesting that compensatory mechanisms may account for maintaining glucose homeostasis. The insulin tolerance tests confirmed improvement of insulin sensitivity accompanied with upregulation of phosphorylated AKT in the peripheral insulin target tissues of TRAPδ βKO mice. Together, these data uncover a critical role of TRAPδ in preproinsulin translocation and insulin biosynthesis. The moderately decreased circulating insulin promotes insulin sensitivity in the insulin target tissues. J.Guo: None. Y.Huang: None. M.Liu: None. Y.Yang: None. N.Xu: None. Y.Yang: None. W.Feng: None. Y.Ye: None. X.Li: None. X.Xu: None. J.Cui: None. National Natural Science Foundation of China (81830025, 82220108014, 82270864, 82070854); National Key R&D Program of China (2019YFA0802502, 2022YFE0131400); Tianjin Municipal Health and Health Committee (TJWJ2021ZD00); Tianjin Key Medical Discipline (Specialty) Construction Project (TJYXZDXK-030A)

Proinsulin-to-C-peptide ratio as a marker of beta-cell function in African American and European American adults

Abstract: <p> </p> <p><strong>Objective</strong>: The primary purpose of the present study was to test the hypothesis that the proinsulin-to-C-peptide ratio (PI:CP), as an index of proinsulin secretion, would be higher and associated with indices of beta-cell function in African American adults relative to European American adults without type 2 diabetes. </p> <p><strong>Research Design and Methods</strong>: Participants were 114 African American and European American adult men and women. A 2-h OGTT was conducted to measure glucose, insulin, C-peptide, and proinsulin, and derive indices of beta-cell response to glucose. The Matsuda index was calculated as a measure of insulin sensitivity. The disposition index (DI, the product of insulin sensitivity and beta-cell response) was calculated for each phase of beta-cell responsivity. Pearson correlations were used to investigate the relationship of PI:CP with each phase of beta-cell response (Φb, Φd, Φs, Φtot), disposition indices (DId, DIs, DItot), and insulin sensitivity. Multiple linear regression analysis was used to evaluate independent contributions of race, BMI, and glucose tolerance status on PI:CP levels, before and after adjustment for insulin sensitivity. </p> <p><strong>Results: </strong>African American participants had higher fasting and 2-h PI:CP. Fasting PI:CP was positively associated with Φb, and fasting PI:CP and 2-h PI:CP were inversely associated with DId and insulin sensitivity only in African American participants. </p> <p><strong>Conclusions: </strong>PI:CP could be useful in identifying African American individuals at highest risk for beta-cell dysfunction and ultimately type 2 diabetes. </p>

Amino acid polymorphisms in human histocompatibility leukocyte antigen class II and proinsulin epitope have impacts on type 1 diabetes mellitus induced by immune-checkpoint inhibitors

Abstract: Introduction Immune-checkpoint inhibitors are effective in various advanced cancers. Type 1 diabetes mellitus induced by them (ICI-T1DM) is a serious complication requiring prompt insulin treatment, but the immunological mechanism behind it is unclear. Methods We examined amino acid polymorphisms in human histocompatibility leukocyte antigen (HLA) molecules and investigated proinsulin epitope binding affinities to HLA molecules. Results and Discussion Twelve patients with ICI-T1DM and 35 patients in a control group without ICI-T1DM were enrolled in the study. Allele and haplotype frequencies of HLA-DRB1*04:05, DQB1*04:01, and most importantly DPB1*05:01 were significantly increased in patients with ICI-T1DM. In addition, novel amino acid polymorphisms in HLA-DR (4 polymorphisms), in DQ (12 polymorphisms), and in DP molecules (9 polymorphisms) were identified. These amino acid polymorphisms might be associated with the development of ICI-T1DM. Moreover, novel human proinsulin epitope clusters in insulin A and B chains were discovered in silico and in vitro peptide binding assays to HLA-DP5. In conclusion, significant amino acid polymorphisms in HLA-class II molecules, and conformational alterations in the peptide-binding groove of the HLA-DP molecules were considered likely to influence the immunogenicity of proinsulin epitopes in ICI-T1DM. These amino acid polymorphisms and HLA-DP5 may be predictive genetic factors for ICI-T1DM.

A proinsulin-dependent interaction between ENPL-1 and ASNA-1 in neurons is required to maintain insulin secretion in C. elegans.

Abstract: Neuropeptides, including insulin, are important regulators of physiological functions of the organisms. Trafficking through the Golgi is crucial for the regulation of secretion of insulin-like peptides. ASNA-1 (TRC40) and ENPL-1 (GRP94) are conserved insulin secretion regulators in Caenorhabditis elegans (and mammals), and mouse Grp94 mutants display type 2 diabetes. ENPL-1/GRP94 binds proinsulin and regulates proinsulin levels in C. elegans and mammalian cells. Here, we have found that ASNA-1 and ENPL-1 cooperate to regulate insulin secretion in worms via a physical interaction that is independent of the insulin-binding site of ENPL-1. The interaction occurs in DAF-28/insulin-expressing neurons and is sensitive to changes in DAF-28 pro-peptide levels. Consistently, ASNA-1 acted in neurons to promote DAF-28/insulin secretion. The chaperone form of ASNA-1 was likely the interaction partner of ENPL-1. Loss of asna-1 disrupted Golgi trafficking pathways. ASNA-1 localization to the Golgi was affected in enpl-1 mutants and ENPL-1 overexpression partially bypassed the ASNA-1 requirement. Taken together, we find a functional interaction between ENPL-1 and ASNA-1 that is necessary to maintain proper insulin secretion in C. elegans and provides insights into how their loss might cause diabetes in mammals.

Single-cell RNAseq identifies clonally expanded antigen-specific T-cells following intradermal injection of gold nanoparticles loaded with diabetes autoantigen in humans

Abstract: Gold nanoparticles (GNPs) have been used in the development of novel therapies as a way of delivery of both stimulatory and tolerogenic peptide cargoes. Here we report that intradermal injection of GNPs loaded with the proinsulin peptide C19-A3, in patients with type 1 diabetes, results in recruitment and retention of immune cells in the skin. These include large numbers of clonally expanded T-cells sharing the same paired T-cell receptors (TCRs) with activated phenotypes, half of which, when the TCRs were re-expressed in a cell-based system, were confirmed to be specific for either GNP or proinsulin. All the identified gold-specific clones were CD8+, whilst proinsulin-specific clones were both CD8+ and CD4+. Proinsulin-specific CD8+ clones had a distinctive cytotoxic phenotype with overexpression of granulysin (GNLY) and KIR receptors. We have demonstrated that the T-cell response to antigen-loaded GNPs injected intradermally is divided between targeting the gold core and the antigenic cargo. In addition, our scRNAseq data indicate that focussing on clonally expanded skin infiltrating T-cells recruited to intradermally injected antigen is a highly efficient method to enrich and identify antigen-specific cells. This approach has the potential to be used to optimise the intradermal delivery of antigens and nanoparticles for immune modulation in humans.