Proinsulin folding and trafficking defects trigger a common pathological disturbance of endoplasmic reticulum homeostasis.

Primary defects in folding of mutant proinsulin can cause dominant-negative proinsulin accumulation in the endoplasmic reticulum (ER), impaired anterograde proinsulin trafficking, perturbed ER homeostasis, diminished insulin production, and ß-cell dysfunction. Conversely, if primary impairment of ER-to-Golgi trafficking (which also perturbs ER homeostasis) drives misfolding of nonmutant proinsulin-this might suggest bi-directional entry into a common pathological phenotype (proinsulin misfolding, perturbed ER homeostasis, and deficient ER export of proinsulin) that can culminate in diminished insulin storage and diabetes. Here, we've challenged ß-cells with conditions that impair ER-to-Golgi trafficking, and devised an accurate means to assess the relative abundance of distinct folded/misfolded forms of proinsulin using a novel nonreducing SDS-PAGE/immunoblotting protocol. We confirm abundant proinsulin misfolding upon introduction of a diabetogenic INS mutation, or in the islets of db/db mice. Whereas blockade of proinsulin trafficking in Golgi/post-Golgi compartments results in intracellular accumulation of properly-folded proinsulin (bearing native disulfide bonds), impairment of ER-to-Golgi trafficking (regardless whether such impairment is achieved by genetic or pharmacologic means) results in decreased native proinsulin with more misfolded proinsulin. Remarkably, reversible ER-to-Golgi transport defects (such as treatment with brefeldin A or cellular energy depletion) upon reversal quickly restore the ER folding environment, resulting in the disappearance of pre-existing misfolded proinsulin while preserving proinsulin bearing native disulfide bonds. Thus, proper homeostatic balance of ER-to-Golgi trafficking is linked to a more favorable proinsulin folding (as well as trafficking) outcome.

Human proinsulin production in the milk of transgenic cattle.

BACKGROUND: The worldwide growing demand for human insulin for treating diabetes could be supplied by transgenic animals producing insulin in their milk. METHODS AND RESULTS: Pseudo-lentivirus containing the bovine ß-casein promoter and human insulin sequences was used to produce modified adult fibroblasts, and the cells were used for nuclear transfer. Transgenic embryos were transferred to recipient cows, and one pregnancy was produced. Recombinant protein in milk was evaluated using western blotting and mass spectrometry. One transgenic cow was generated, and in milk analysis, two bands were observed in western blotting with a molecular mass corresponding to the proinsulin and insulin. The mass spectrometry analysis showed the presence of human insulin more than proinsulin in the milk, and it identified proteases in the transgenic milk that could convert proinsulin into insulin and insulin-degrading enzyme that could degrade the recombinant protein. CONCLUSION: The methodologies used for generating the transgenic cow allowed the detection of the production of recombinant protein in the milk at low relative expression compared to milk proteins, using mass spectrometry, which was efficient for detecting recombinant protein with low expression in milk. Milk proteases could act on protein processing converting recombinant protein to functional protein. On the other hand, some milk proteases could act in degrading the recombinant protein.

Osteocalcin protects islet identity in low-density lipoprotein receptor knockout mice on high-fat diet.

Metabolic syndrome (MetS) is an increasing global health threat and strong risk factor for type 2 diabetes (T2D). MetS causes both hyperinsulinemia and islet size overexpansion, and pancreatic ß-cell failure impacts insulin and proinsulin secretion, mitochondrial density, and cellular identity loss. The low-density lipoprotein receptor knockout (LDLr-/-) model combined with high-fat diet (HFD) has been used to study alterations in multiple organs, but little is known about the changes to ß-cell identity resulting from MetS. Osteocalcin (OC), an insulin-sensitizing protein secreted by bone, shows promising impact on ß-cell identity and function. LDLr-/- mice at 12 months were fed chow or HFD for 3 months ± 4.5 ng/h OC. Islets were examined by immunofluorescence for alterations in nuclear Nkx6.1 and PDX1 presence, insulin-glucagon colocalization, islet size and %ß-cell and islet area by insulin and synaptophysin, and mitochondria fluorescence intensity by Tomm20. Bone mineral density (BMD) and %fat changes were examined by Piximus Dexa scanning. HFD-fed mice showed fasting hyperglycemia by 15 months, increased weight gain, %fat, and fasting serum insulin and proinsulin; concurrent OC treatment mitigated weight increase and showed lower proinsulin-to-insulin ratio, and higher BMD. HFD increased %ß and %islet area, while simultaneous OC-treatment with HFD was comparable to chow-fed mice. Significant reductions in nuclear PDX1 and Nkx6.1 expression, increased insulin-glucagon colocalization, and reduction in ß-cell mitochondria fluorescence intensity were noted with HFD, but largely prevented with OC administration. OC supplementation here suggests a benefit to ß-cell identity in LDLr-/- mice and offers intriguing clinical implications for countering metabolic syndrome.

Insulin resistance prior to term age in very low birthweight infants: a prospective study.

OBJECTIVE: To explore the glucose-related hormone profile of very low birthweight (VLBW) infants and assess the association between neonatal hyperglycaemia and insulin resistance during the admission period. DESIGN: A prospective observational study-the Very Low Birth Weight Infants, Glucose and Hormonal Profiles over Time study. SETTING: A tertiary neonatal intensive care unit and four neonatal units in county hospitals in Sweden. PATIENTS: 48 infants born <1500 g (VLBW) during 2016-2019. OUTCOME MEASURES: Plasma concentrations of glucose-related hormones and proteins (C-peptide, insulin, ghrelin, glucagon-like peptide 1 (GLP-1), glucagon, leptin, resistin and proinsulin), insulin:C-peptide and proinsulin:insulin ratios, Homoeostatic Model Assessment 2 (HOMA2) and Quantitative Insulin Sensitivity Check (QUICKI) indices, measured on day of life (DOL) 7 and at postmenstrual age 36 weeks. RESULTS: Lower gestational age was significantly associated with higher glucose, C-peptide, insulin, proinsulin, leptin, ghrelin, resistin and GLP-1 concentrations, increased HOMA2 index, and decreased QUICKI index and proinsulin:insulin ratio. Hyperglycaemic infants had significantly higher glucose, C-peptide, insulin, leptin and proinsulin concentrations, and lower QUICKI index, than normoglycaemic infants. Higher glucose and proinsulin concentrations and insulin:C-peptide ratio, and lower QUICKI index on DOL 7 were significantly associated with longer duration of hyperglycaemia during the admission period. CONCLUSIONS: VLBW infants seem to have a hormone profile consistent with insulin resistance. Lower gestational age and hyperglycaemia are associated with higher concentrations of insulin resistance markers.

Permanent neonatal diabetes-causing insulin mutations have dominant negative effects on beta cell identity.

OBJECTIVE: Heterozygous coding sequence mutations of the INS gene are a cause of permanent neonatal diabetes (PNDM), requiring insulin therapy similar to T1D. While the negative effects on insulin processing and secretion are known, how dominant insulin mutations result in a continued decline of beta cell function after birth is not well understood. METHODS: We explored the causes of beta cell failure in two PNDM patients with two distinct INS mutations using patient-derived iPSCs and mutated hESCs. RESULTS: we detected accumulation of misfolded proinsulin and impaired proinsulin processing in vitro, and a dominant-negative effect of these mutations on beta-cell mass and function after transplantation into mice. In addition to anticipated ER stress, we found evidence of beta-cell dedifferentiation, characterized by an increase of cells expressing both Nkx6.1 and ALDH1A3, but negative for insulin and glucagon. CONCLUSIONS: These results highlight a novel mechanism, the loss of beta cell identity, contributing to the loss and functional failure of human beta cells with specific insulin gene mutations.

Loss of the Golgi-localized v-ATPase subunit does not alter insulin granule formation or pancreatic islet ß-cell function.

Delayed Golgi export of proinsulin has recently been identified as an underlying mechanism leading to insulin granule loss and ß-cell secretory defects in type 2 diabetes (T2D). Because acidification of the Golgi lumen is critical for proinsulin sorting and delivery into the budding secretory granule, we reasoned that dysregulation of Golgi pH may contribute to proinsulin trafficking defects. In this report, we examined pH regulation of the Golgi and identified a partial alkalinization of the Golgi lumen in a diabetes model. To further explore this, we generated a ß-cell specific knockout (KO) of the v0a2 subunit of the v-ATPase pump, which anchors the v-ATPase to the Golgi membrane. Although loss of v0a2 partially neutralized Golgi pH and was accompanied by distension of the Golgi cisternae, proinsulin export from the Golgi and insulin granule formation were not affected. Furthermore, ß-cell function was well preserved. ß-cell v0a2 KO mice exhibited normal glucose tolerance in both sexes, no genotypic difference to diet-induced obesity, and normal insulin secretory responses. Collectively, our data demonstrate the v0a2 subunit contributes to ß-cell Golgi pH regulation but suggest that additional disturbances to Golgi structure and function contribute to proinsulin trafficking defects in diabetes.NEW & NOTEWORTHY Delayed proinsulin export from the Golgi in diabetic ß-cells contributes to decreased insulin granule formation, but the underlying mechanisms are not clear. Here, we explored if dysregulation of Golgi pH can alter Golgi function using ß-cell specific knockout (KO) of the Golgi-localized subunit of the v-ATPase, v0a2. We show that partial alkalinization of the Golgi dilates the cisternae, but does not affect proinsulin export, insulin granule formation, insulin secretion, or glucose homeostasis.

An Insulin-Chromogranin A Hybrid Peptide Activates DR11-Restricted T Cells in Human Type 1 Diabetes.

Hybrid insulin peptides (HIPs) formed through covalent cross-linking of proinsulin fragments to secretory granule peptides are detectable within murine and human islets. The 2.5HIP (C-peptide-chromogranin A [CgA] HIP), recognized by the diabetogenic BDC-2.5 clone, is a major autoantigen in the nonobese diabetic mouse. However, the relevance of this epitope in human disease is currently unclear. A recent study probed T-cell reactivity toward HIPs in patients with type 1 diabetes, documenting responses in one-third of the patients and isolating several HIP-reactive T-cell clones. In this study, we isolated a novel T-cell clone and showed that it responds vigorously to the human equivalent of the 2.5HIP (designated HIP9). Although the responding patient carried the risk-associated DRB1*04:01/DQ8 haplotype, the response was restricted by DRB1*11:03 (DR11). HLA class II tetramer staining revealed higher frequencies of HIP9-reactive T cells in individuals with diabetes than in control participants. Furthermore, in DR11+ participants carrying the DRB4 allele, HIP9-reactive T-cell frequencies were higher than observed frequencies for the immunodominant proinsulin 9-28 epitope. Finally, there was a negative correlation between HIP9-reactive T-cell frequency and age at diagnosis. These results provide direct evidence that this C-peptide-CgA HIP is relevant in human type 1 diabetes and suggest a mechanism by which nonrisk HLA haplotypes may contribute to the development of ß-cell autoimmunity.

Independent activation of CREB3L2 by glucose fills a regulatory gap in mouse ß-cells by co-ordinating insulin biosynthesis with secretory granule formation.

OBJECTIVE: Although individual steps have been characterized, there is little understanding of the overall process whereby glucose co-ordinates the biosynthesis of insulin with its export out of the endoplasmic reticulum (ER) and incorporation into insulin secretory granules (ISGs). Here we investigate a role for the transcription factor CREB3L2 in this context. METHODS: MIN6 cells and mouse islets were analysed by immunoblotting after treatment with glucose, fatty acids, thapsigargin and various inhibitors. Knockdown of CREB3L2 was achieved using si or sh constructs by transfection, or viral delivery. In vivo metabolic phenotyping was conducted after deletion of CREB3L2 in ß-cells of adult mice using Ins1-CreER+. Islets were isolated for RNAseq and assays of glucose-stimulated insulin secretion (GSIS). Trafficking was monitored in islet monolayers using a GFP-tagged proinsulin construct that allows for synchronised release from the ER. RESULTS: With a Km ≈3.5 mM, glucose rapidly (T1/2 0.9 h) increased full length (FL) CREB3L2 followed by a slower rise (T1/2 2.5 h) in its transcriptionally-active cleavage product, P60 CREB3L2. Glucose stimulation repressed the ER stress marker, CHOP, and this was partially reverted by knockdown of CREB3L2. Activation of CREB3L2 by glucose was not due to ER stress, however, but a combination of O-GlcNAcylation, which impaired proteasomal degradation of FL-CREB3L2, and mTORC1 stimulation, which enhanced its conversion to P60. cAMP generation also activated CREB3L2, but independently of glucose. Deletion of CREB3L2 inhibited GSIS ex vivo and, following a high-fat diet (HFD), impaired glucose tolerance and insulin secretion in vivo. RNAseq revealed that CREB3L2 regulated genes controlling trafficking to-and-from the Golgi, as well as a broader cohort associated with ß-cell compensation during a HFD. Although post-Golgi trafficking appeared intact, knockdown of CREB3L2 impaired the generation of both nascent ISGs and proinsulin condensates in the Golgi, implying a defect in ER export of proinsulin and/or its processing in the Golgi. CONCLUSION: The stimulation of CREB3L2 by glucose defines a novel, rapid and direct mechanism for co-ordinating the synthesis, packaging and storage of insulin, thereby minimizing ER overload and optimizing ß-cell function under conditions of high secretory demand. Upregulation of CREB3L2 also potentially contributes to the benefits of GLP1 agonism and might in itself constitute a novel means of treating ß-cell failure.

Association of common variants of TCF7L2 and PCSK2 with gestational diabetes mellitus in West Bengal, India.

The genetic etiology of gestational diabetes mellitus (GDM) was suggested to overlap with type-2 diabetes(T2D). Transcription factor 7-like 2 (TCF7L2) and Proprotein Convertase Subtilisin/Kexin type 2 (PCSK2) are T2D susceptibility genes of the insulin synthesis/processing pathway. We analyzed associations of TCF7L2 and PCSK2 variants with GDM risk and evaluated their potential impact on impaired insulin processing in an eastern Indian population. The study included 114 GDM (case) and 228 non-GDM pregnant women (control). rs7903146, rs4132670, rs12255372 of TCF7L2, and rs2269023 of PCSK2 were genotyped by PCR-RFLP, and genotype distributions were compared between case and control. Fasting serum proinsulin and C-peptide levels were measured by ELISA and the Proinsulin/C-peptide ratio was considered an indicator of proinsulin conversion. Significantly higher frequency of risk allele (T) of rs12255372 (p = 0.02, OR = 2.0, 95%CI = 1.11-3.64) and rs4132670 (p = 0.002, OR = 2.26, 95%CI = 1.32-3.87) of TCF7L2 was found in GDM cases than non-GDM controls; TT genotype was associated with significantly increased disease risk. In rs7903146 (TCF7L2) and rs2269023 (PCSK2), although the frequency of risk allele (T) was not significantly higher in cases than controls, an association of TT for both variants remained significant with higher GDM risk in the recessive model. Increased serum pro-insulin and proinsulin:c-peptide ratio was found in GDM than non-GDM women and the phenomenon showed significant association with careers of risk alleles for TCF7L2 variants. In conclusion, TCF7L2 and PCSK2 variants are related to GDM risk in the studied population and hence may serve as potential biomarkers for assessing the disease risk. TCF7L2 variants contribute to impaired insulin processing.

Immunohistochemical and in situ hybridization analyses of glucose transporter 2 in pancreatic neuroendocrine tumors: Possible glucose transporter 2 association with neoplastic insulin production.

BACKGROUND: Pancreatic neuroendocrine tumors (PanNETs) are rare neoplasms. Additionally, glucose transporter 2 (GLUT2) is associated with insulin production and is essential for glucose transport to normal pancreatic ß-cells. Neoplastic cell GLUT2 expression may also influence insulin production by using this transporter. GLUT2 expression and its clinical significance remain unclear in PanNETs. This study aimed to provide GLUT2 expression profiles and evidence of correlation with insulin in PanNETs. METHODS: Clinical data were retrieved from 113 surgically resected paraffin-embedded PanNET tissue samples fixed with 10% formalin. PanNETs are categorized as insulinoma, non-functional (NF)-PanNET, or PanNET-not otherwise specified (NOS). A GLUT2 score was used to evaluate cytoplasmic GLUT2 immunoreactivity. The immunoreactive score (IRS) was used to determine membranous GLUT2, cytoplasmic insulin, and proinsulin immunoreactivities. A commercially available in situ hybridization (ISH) kit detected human SLC2A2 (GLUT2) mRNA on tissues in all seven positive- and 20 negative-GLUT2 IRS cases. RESULTS: GLUT2 and IRSs significantly differed among insulinoma, NF-PanNET, and PanNET-NOS. Insulinomas exhibited significantly higher GLUT2 scores and IRSs than did NF-PanNETs. GLUT2 IRS positive cases demonstrated significantly higher insulin and proinsulin IRSs than did negative cases. Additionally, GLUT2 ISH-positive cases demonstrated positive GLUT2 scores and IRSs, with significantly higher GLUT2 IRSs than did negative cases. PanNET histological grade categories did not significantly affect GLUT2 scores and IRSs. CONCLUSION: The first evidence of a correlation between GLUT2 expressions and insulin in PanNETs is shown in this study.

Non-cross-linking advanced glycation end products affect prohormone processing.

Advanced glycation end products (AGEs) are non-enzymatic post-translational modifications of amino acids and are associated with diabetic complications. One proposed pathomechanism is the impaired processing of AGE-modified proteins or peptides including prohormones. Two approaches were applied to investigate whether substrate modification with AGEs affects the processing of substrates like prohormones to the active hormones. First, we employed solid-phase peptide synthesis to generate unmodified as well as AGE-modified protease substrates. Activity of proteases towards these substrates was quantified. Second, we tested the effect of AGE-modified proinsulin on the processing to insulin. Proteases showed the expected activity towards the unmodified peptide substrates containing arginine or lysine at the C-terminal cleavage site. Indeed, modification with Nε-carboxymethyllysine (CML) or methylglyoxal-hydroimidazolone 1 (MG-H1) affected all proteases tested. Cysteine cathepsins displayed a reduction in activity by ∼50% towards CML and MG-H1 modified substrates. The specific proteases trypsin, proprotein convertases subtilisin-kexins (PCSKs) type proteases, and carboxypeptidase E (CPE) were completely inactive towards modified substrates. Proinsulin incubation with methylglyoxal at physiological concentrations for 24 h resulted in the formation of MG-modified proinsulin. The formation of insulin was reduced by up to 80% in a concentration-dependent manner. Here, we demonstrate the inhibitory effect of substrate-AGE modifications on proteases. The finding that PCSKs and CPE, which are essential for prohormone processing, are inactive towards modified substrates could point to a yet unrecognized pathomechanism resulting from AGE modification relevant for the etiopathogenesis of diabetes and the development of obesity.

A first-in-human, open-label Phase 1b and a randomised, double-blind Phase 2a clinical trial in recent-onset type 1 diabetes with AG019 as monotherapy and in combination with teplizumab.

AIMS/HYPOTHESIS: We hypothesised that islet beta cell antigen presentation in the gut along with a tolerising cytokine would lead to antigen-specific tolerance in type 1 diabetes. We evaluated this in a parallel open-label Phase 1b study using oral AG019, food-grade Lactococcus lactis bacteria genetically modified to express human proinsulin and human IL-10, as a monotherapy and in a parallel, randomised, double-blind Phase 2a study using AG019 in combination with teplizumab. METHODS: Adults (18-42 years) and adolescents (12-17 years) with type 1 diabetes diagnosed within 150 days were enrolled, with documented evidence of at least one autoantibody and a stimulated peak C-peptide level >0.2 nmol/l. Participants were allocated to interventions using interactive response technology. We treated 42 people aged 12-42 years with recent-onset type 1 diabetes, 24 with Phase 1b monotherapy (open-label) and 18 with Phase 2a combination therapy. In the Phase 2a study, after treatment of the first two open-label participants, all people involved were blinded to group assignment, except for the Data Safety Monitoring Board members and the unblinded statistician. The primary endpoint was safety and tolerability based on the incidence of treatment-emergent adverse events, collected up to 6 months post treatment initiation. The secondary endpoints were pharmacokinetics, based on AG019 detection in blood and faeces, and pharmacodynamic activity. Metabolic and immune endpoints included stimulated C-peptide levels during a mixed meal tolerance test, HbA1c levels, insulin use, and antigen-specific CD4+ and CD8+ T cell responses using an activation-induced marker assay and pooled tetramers, respectively. RESULTS: Data from 24 Phase 1b participants and 18 Phase 2a participants were analysed. No serious adverse events were reported and none of the participants discontinued AG019 due to treatment-emergent adverse events. No systemic exposure to AG019 bacteria, proinsulin or human IL-10 was demonstrated. In AG019 monotherapy-treated adults, metabolic variables were stabilised up to 6 months (C-peptide, insulin use) or 12 months (HbA1c) post treatment initiation. In participants treated with AG019/teplizumab combination therapy, all measured metabolic variables stabilised or improved up to 12 months and CD8+ T cells with a partially exhausted phenotype were significantly increased at 6 months. Circulating preproinsulin-specific CD4+ and CD8+ T cells were detected before and after treatment, with a reduction in the frequency of preproinsulin-specific CD8+ T cells after treatment with monotherapy or combination therapy. CONCLUSIONS/INTERPRETATION: Oral delivery of AG019 was well tolerated and safe as monotherapy and in combination with teplizumab. AG019 was not shown to interfere with the safety profile of teplizumab and may have additional biological effects, including changes in preproinsulin-specific T cells. These preliminary data support continuing studies with this agent alone and in combination with teplizumab or other systemic immunotherapies in type 1 diabetes. TRIAL REGISTRATION: ClinicalTrials.gov NCT03751007, EudraCT 2017-002871-24 FUNDING: This study was funded by Precigen ActoBio.

Positive association between proinsulin and fatty liver index in people with type 2 diabetes.

The post-hoc study, derived from our previous prospective observational study, investigated the association between fasting serum proinsulin levels and hepatic steatosis in people with type 2 diabetes. The severity of hepatic steatosis was assessed using the fatty liver index. A total of 268 participants were divided into three groups: low (n = 110), moderate (n = 75), and high fatty liver index (n = 83). In both the crude and age/sex-adjusted analysis, logarithm-transformed proinsulin was significantly higher in the high fatty liver index group than in the low or moderate groups (all p < 0.01). The moderate fatty liver index group showed higher logarithm-transformed proinsulin than the low group (both p < 0.01). Positive associations between proinsulin and fatty liver index shown in this study would support an involvement of hepato-pancreatic crosstalk in the pathophysiology of type 2 diabetes.

Serum proinsulin levels as peripheral blood biomarkers in patients with cognitive impairment.

Insulin has long been associated with dementia. Insulin affecting the clearance of amyloid-ß peptide and phosphorylation of tau in the CNS. Proinsulin is a precursor of insulin and its elevated serum levels are associated with peripheral insulin resistance that may reduce brain insulin levels. Our study aimed to assess differences in serum proinsulin levels between normal and cognitive impairment groups. Prospective recruitment of elderly participants was initiated from October 2019 to September 2023. Patients were divided into "cognitive impairment" and "normal cognition" group. All participants had blood drawn and serum proinsulin was measured at baseline and 12 months. Neurocognitive testing was performed every 6 months. A total of 121 participants were recruited. Seventy-seven were in the normal cognition group and 44 in the cognitive impairment group. The glycemic control and prevalence of diabetes type 2 was similar between groups. Baseline serum proinsulin levels were higher in the cognitively impaired group compared to the normal group at baseline (p = 0.019) and correlated with worse cognitive scores. We identified cognitive status, age, and BMI as potential factors associated with variations in baseline proinsulin levels. Given the complex interplay between insulin and dementia pathogenesis, serum biomarkers related to insulin metabolism may exhibit abnormalities in cognitive impaired patients. Here we present the proinsulin levels in individuals with normal cognitive function versus those with cognitive impairment and found a significant difference. This observation may help identifying non-diabetic patients suitable for treatment with novel AD drugs that related to insulin pathway.

Age-Related Changes in Proinsulin Processing in Normoglycemic Individuals.

In order to understand the pathological changes associated with glucose homeostasis in old age, it is necessary to know the natural changes in the processing of proinsulin to mature insulin. While there is abundant information about insulin production and function in diabetics, the situation in healthy adults and the elderly has surprisingly rarely been investigated. The aim of the study was to determine how proinsulin secretion changes in individuals with normal glucose tolerance during the process of natural aging. A total of 761 individuals (539 women, 222 men) aged 18-90 years with normal fasting glycemia (less than 5.6 mmol/l) were divided into five groups according to age. Body composition and levels of fasting blood glucose, proinsulin, insulin, and C-peptide were determined, and the ratios of proinsulin to both insulin and C-peptide were calculated. The homeostasis model of ?-cell function (HOMA F) and peripheral insulin resistance (HOMA R) were calculated. The effect of age was assessed using an ANOVA model consisting of the factors sex, age, and sex × age interaction. Statgraphics Centurion v. XVIII statistical software was used. Glycemia, insulin, C-peptide and HOMA R increased in both sexes up to 75 years. On the contrary, proinsulin levels as well as proinsulin/insulin and proinsulin/C-peptide ratios decreased with age up to 75 years. In normoglycemic and normotolerant people, both women and men, the aging process is associated with decreased insulin sensitivity compensated by potentiation of insulin production. In older age, there is also a gradual decrease in circulating proinsulin, which can be explained by its more efficient processing into active insulin by matured healthy beta cells.

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

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. Clonally expanded antigen-specific T cells remained in situ for months to years, with a spectrum of tissue resident memory and effector memory phenotypes. As the T-cell response is divided between targeting the gold core and the antigenic cargo, this offers a route to improving resident memory T-cells formation in response to vaccines. In addition, our scRNAseq data indicate that focusing 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 monitor the intradermal delivery of antigens and nanoparticles for immune modulation in humans.

Top-Down Proteomics of Mouse Islets With Beta Cell CPE Deletion Reveals Molecular Details in Prohormone Processing.

Altered prohormone processing, such as with proinsulin and pro-islet amyloid polypeptide (proIAPP), has been reported as an important feature of prediabetes and diabetes. Proinsulin processing includes removal of several C-terminal basic amino acids and is performed principally by the exopeptidase carboxypeptidase E (CPE), and mutations in CPE or other prohormone convertase enzymes (PC1/3 and PC2) result in hyperproinsulinemia. A comprehensive characterization of the forms and quantities of improperly processed insulin and other hormone products following Cpe deletion in pancreatic islets has yet to be attempted. In the present study we applied top-down proteomics to globally evaluate the numerous proteoforms of hormone processing intermediates in a ß-cell-specific Cpe knockout mouse model. Increases in dibasic residue-containing proinsulin and other novel proteoforms of improperly processed proinsulin were found, and we could classify several processed proteoforms as novel substrates of CPE. Interestingly, some other known substrates of CPE remained unaffected despite its deletion, implying that paralogous processing enzymes such as carboxypeptidase D (CPD) can compensate for CPE loss and maintain near normal levels of hormone processing. In summary, our quantitative results from top-down proteomics of islets provide unique insights into the complexity of hormone processing products and the regulatory mechanisms.

Adolescents with obesity treated with exenatide maintain endogenous GLP-1, reduce DPP-4, and improve glycemic control.

Background: GLP-1 receptor agonists (GLP-1RA) are increasingly used to treat adolescent obesity. However, the effect on endogenous GLP-1 secretory patterns following treatment in adolescents is unknown. The GLP-1RA exenatide was shown to significantly lower BMI and 2-hour glucose in adolescents with obesity, in the placebo-controlled, randomized controlled trial Combat-JUDO. The aim of this study was to evaluate effects of weekly injections of 2 mg exenatide extended release on secretory patterns of endogenous hormones during OGTT. Subjects and Measurements: This study was a pre-planned sub-study of the Combat-JUDO trial, set at the Pediatric clinic at Uppsala University Hospital, Sweden and Paracelsus Medical University, Austria. 44 adolescents with obesity were included and randomized 1:1 to treatment:placebo. 19 patients in the treatment group and 18 in the placebo group completed the trial. Before and after treatment, GLP-1, glucose, insulin, glucagon and glicentin levels were measured during OGTT; DPP-4 and proinsulin were measured at fasting. A per-protocol approach was used in the analyses. Results: Exenatide treatment did not affect GLP-1 levels during OGTT. Treatment significantly lowered DPP-4, proinsulin and the proinsulin-to-insulin ratio at fasting, increased glicentin levels but did not affect insulin, C-peptide or glucagon levels during OGTT. Conclusion: Weekly s.c. injections with 2 mg of exenatide maintains endogenous total GLP-1 levels and lowers circulating DPP-4 levels. This adds an argument in favor of using exenatide in the treatment of pediatric obesity. Clinical trial registration: clinicaltrials.gov, identifier NCT02794402.

Baseline plasma proinsulin response to glucose for predicting therapeutic response to otelixizumab in recent-onset type 1 diabetes.

AIMS: In recent-onset type 1 diabetes, clamp-derived C-peptide predicts good response to anti-CD3. Elevated proinsulin and proinsulin/C-peptide ratio (PI/CP) suggest increased metabolic/inflammatory beta cell burden. We reanalyzed trial data to compare the ability of baseline acutely glucose-stimulated proinsulin, C-peptide and PI/CP to predict functional outcome. METHODS: Eighty recent-onset type 1 diabetes patients participated in the placebo-controlled otelixizumab (GSK; NCT00627146) trial. Hyperglycemic clamps were performed at baseline, 6, 12 and 18 months, involving 3 h of induced euglycemia, followed by acutely raising and maintaining glycemia to ≥ 10 mmol/l for 140 min. Plasma proinsulin, C-peptide and PI/CP were determined after acute (minute 0 at 10 mmol/l; PI0, CP0, PI/CP0) and sustained glucose stimulation (AUC between minutes 60-140). Outcome was assessed as change in AUC60-140 C-peptide from baseline. RESULTS: In multiple linear regression, higher baseline (≥median [P50]) PI0 independently predicted preservation of beta cell function in response to anti-CD3 and interacted significantly with IAA. During follow-up, anti-CD3 tempered a further increase in PI/CP0, but not in PI0. CP0 outperformed PI0 and PI/CP0 for post-treatment monitoring. CONCLUSIONS: In recent-onset type 1 diabetes, elevated acutely glucose-stimulated proinsulin may complement or replace acutely or sustainedly stimulated C-peptide release for identifying good responders to anti-CD3, but not as outcome measure.

Characterization of impaired beta and alpha cell function in response to an oral glucose challenge in cystic fibrosis: a cross-sectional study.

Aims: The purpose of the study was to further elucidate the pathophysiology of cystic fibrosis (CF)-related diabetes (CFRD) and potential drivers of hypoglycaemia. Hence, we aimed to describe and compare beta cell function (insulin and proinsulin) and alpha cell function (glucagon) in relation to glucose tolerance in adults with CF and to study whether hypoglycaemia following oral glucose challenge may represent an early sign of islet cell impairment. Methods: Adults with CF (≥18 years) were included in a cross-sectional study using an extended (-10, -1, 10, 20, 30, 45, 60, 90, 120, 150, and 180 min) or a standard (-1, 30, 60, and 120 min) oral glucose tolerance test (OGTT). Participants were classified according to glucose tolerance status and hypoglycaemia was defined as 3-hour glucose <3.9 mmol/L in those with normal glucose tolerance (NGT) and early glucose intolerance (EGI). Results: Among 93 participants, 67 underwent an extended OGTT. In addition to worsening in insulin secretion, the progression to CFRD was associated with signs of beta cell stress, as the fasting proinsulin-to-insulin ratio incrementally increased (p-value for trend=0.013). The maximum proinsulin level (pmol/L) was positively associated with the nadir glucagon, as nadir glucagon increased 6.2% (95% confidence interval: 1.4-11.3%) for each unit increase in proinsulin. Those with hypoglycaemia had higher 60-min glucose, 120-min C-peptide, and 180-min glucagon levels (27.8% [11.3-46.7%], 42.9% [5.9-92.85%], and 80.3% [14.9-182.9%], respectively) and unaltered proinsulin-to-insulin ratio compared to those without hypoglycaemia. Conclusions: The maximum proinsulin concentration was positively associated with nadir glucagon during the OGTT, suggesting that beta cell stress is associated with abnormal alpha cell function in adults with CF. In addition, hypoglycaemia seemed to be explained by a temporal mismatch between glucose and insulin levels rather than by an impaired glucagon response.