Insulin Precursor Research Articles

Proinsulin degradation and presentation of a proinsulin B-chain autoantigen involves ER-associated protein degradation (ERAD)-enzyme UBE2G2.

Type 1 diabetes (T1D) is characterized by HLA class I-mediated presentation of autoantigens on the surface of pancreatic β-cells. Recognition of these autoantigens by CD8+ T cells results in the destruction of pancreatic β-cells and, consequently, insulin deficiency. Most epitopes presented at the surface of β-cells derive from the insulin precursor molecule proinsulin. The intracellular processing pathway(s) involved in the generation of these peptides are poorly defined. In this study, we show that a proinsulin B-chain antigen (PPIB5-14) originates from proinsulin molecules that are processed by ER-associated protein degradation (ERAD) and thus originate from ER-resident proteins. Furthermore, screening genes encoding for E2 ubiquitin conjugating enzymes, we identified UBE2G2 to be involved in proinsulin degradation and subsequent presentation of the PPIB10-18 autoantigen. These insights into the pathway involved in the generation of insulin-derived peptides emphasize the importance of proinsulin processing in the ER to T1D pathogenesis and identify novel targets for future T1D therapies.

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-β 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.

Expression of recombinant human insulin precursor by Pichia pastoris in a 10 liter bioreactor

Recombinant insulin is a vital medicine for diabetic patients. This hormone is produced by microbes such as Pichia pastoris that carry the recombinant gene of a human insulin precursor (HIP). Large-scale protein production involves a bioreactor to promote the optimal condition for the yeast to express the protein target. In order to obtain a large amount of insulin, the cultivation of recombinant P.pastoris/pD902-IP carrying human insulin precursor gene in a bioreactor 10 Liter was developed. The isolate was cultivated in a half concentration of basal salt media for 124.5 hours. Induction of the protein was done by continual methanol feeding. The fermentation condition was set to have a temperature at 28°C, agitation at 300 rpm, aeration at 2 L/min and a pH value of around 5. Dry cell weight (DCW) was measured, and HPLC quantified the content of HIP, glycerol and methanol. This work’s DCW and HIP concentrations were 46.5 g/L and 928 mg/L, respectively. The results can be higher by increasing the number of cells in the culture or extending the cultivation time so that the HIP concentration may exceed 1 g/L.

Simultaneous Administration of Berberine and Transplantation of Endometrial Stem Cell-Derived Insulin Precursor Cells on a Nanofibrous Scaffold to Treat Diabetes Mellitus in Mice.

Today, significant success has been achieved in treating diabetes with cell therapy derived from various sources of stem and progenitors. The replacement of beta cells is one of the new diabetes treatment methods. To this end, the production of pancreatic beta precursors in cell culture has created an important research field for diabetes treatment. Endometrial stem cells were isolated using an enzymatic method, and after their identity was confirmed using a flow cytometry and differentiation potential assay, the isolated cells were cultured on an electrospun PCL/CS scaffold. Endometrial cells were differentiated into insulin-producing cells (IPCs), and gene expression was analyzed using the qRT-PCR and immunofluorescence to confirm the creation of IPCs. Then, IPCs on the scaffold along with berberine were applied to 5 groups of diabetic mice, and after 6 weeks, insulin, blood glucose, and weight of the animals were measured. The findings revealed that pancreatic markers were significantly expressed in IPCs compared to control cells. In addition, when compared to the control group and scaffolds, the receiving group of IPCs on scaffolds had a significant improvement (p ≤ 0.0015), and this improvement increased with the addition of berberine (decrease in blood sugar (133 mg/dL), and an increase in weight (5/39 g) and insulin (2.29 MIU/L). Thus, tissue engineering is a promising new strategy for treating diabetes and can be used in the future for cell therapy and suitable drugs for diabetic patients.

Antagonistic Insulin Derivative Suppresses Insulin-Induced Hypoglycemia.

Insulin derivatives provide new functions that are distinctive from native insulin. We investigated insulin modifications on the C-terminal A chain with insulin receptor (IR) peptide binders and presented a full and potent IR antagonist. We prepared insulin precursors featuring a sortase A (SrtA) recognition sequence, LPETGG, at the C-terminal A chain and used a SrtA-mediated ligation method to synthesize insulin derivatives. The insulin precursor exhibits full IR agonism potency, similar to native human insulin. We explored derivatives with linear IR binding peptides attached to the insulin C-terminal A chain. One insulin derivative with an IR binder (Ins-AC-S2) can fully antagonize IR activation by insulin, as confirmed by cell-based assays. This IR antagonist suppresses insulin-induced hypoglycemia in a streptozotocin-induced diabetic rat model. This study provides a new direction toward insulin antagonist development.

Full-length versus truncated α-factor secretory signal sequences for expression of recombinant human insulin precursor in yeast Pichia pastoris: a comparison

BackgroundHuman insulin was the first FDA-approved biopharmaceutical drug produced through recombinant DNA technology. The previous studies successfully expressed recombinant human insulin precursors (HIP) in Pichia pastoris truncated and full-length α-factor recombinant clones. The matting α-factor (Matα), a signal secretion, direct the HIP protein into the culture media. This study aimed to compare the HIP expression from full-length and truncated α-factor secretory signals clones that grown in two types of media, buffered methanol complex medium (BMMY) and methanol basal salt medium (BSMM). ResultsImageJ analysis of the HIP’s SDS-PAGE shows that the average HIP expression level of the recombinant P. pastoris truncated α-factor clone (CL4) was significantly higher compared to the full-length (HF7) when expressed in both media. Western blot analysis showed that the expressed protein was the HIP. The α-factor protein structure was predicted using the AlphaFold and visualized using UCSF ChimeraX to confirm the secretion ability for both clones. ConclusionsCL4 clone, which utilized a truncated α-factor in the P. pastoris HIP expression cassette, significantly expressed HIP 8.97 times (in BMMY) and 1.17 times (in BSMM) higher than HF7 clone, which used a full-length α-factor secretory signal. This research confirmed that deletion of some regions of the secretory signal sequence significantly improved the efficiency of HIP protein expression in P. pastoris.

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

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.

Development of human insulin precursor expressed from P.pastoris-X33 in a 10L-fermenter

Human insulin is a hormone that regulate blood glucose in human. Due to the vital role of insulin and the trend of diabetic case globally, insulin demand has been rise in the world. Indonesia as a country that had diabetic patient more than 10 million people in 2017 is still imported raw material of human insulin. The development of production process of human insulin is needed in order to support government objective to be independence in medicine aspect. Human insulin precursor (HIP) expressed from a Pichia pastoris X33/pD902-IP had been developed and optimised in a small scale. However, the scaling up in fermenter 10L has not been studied. Using a 10L-fermenter the fermentation system of P.pastoris X33/pD902-IP was developed. Fermentation done in 120 hours using a basal salt medium (half concentration) for the vegetative and induction media. To induce HIP expression, methanol is fed by pulse strategy with a gradient concentration 1-3% for 48 hours. The dry cell weight (DCW) and HIP titer were 72 g/L and 192 mg/L, respectively. This development is the first fermentation process of HIP in fermenter 10L in Indonesia.

Inflammatory Cytokines Rewire the Proinsulin Interaction Network in Human Islets.

Aberrant biosynthesis and secretion of the insulin precursor proinsulin occurs in both type I and type II diabetes. Inflammatory cytokines are implicated in pancreatic islet stress and dysfunction in both forms of diabetes, but the mechanisms remain unclear. We sought to determine the effect of the diabetes-associated cytokines on proinsulin folding, trafficking, secretion, and β-cell function. Human islets were treated with interleukin-1β and interferon-γ for 48 hours, followed by analysis of interleukin-6, nitrite, proinsulin and insulin release, RNA sequencing, and unbiased profiling of the proinsulin interactome by affinity purification-mass spectrometry. Cytokine treatment induced secretion of interleukin-6, nitrites, and insulin, as well as aberrant release of proinsulin. RNA sequencing showed that cytokines upregulated genes involved in endoplasmic reticulum stress, and, consistent with this, affinity purification-mass spectrometry revealed cytokine induced proinsulin binding to multiple endoplasmic reticulum chaperones and oxidoreductases. Moreover, increased binding to the chaperone immunoglobulin binding protein was required to maintain proper proinsulin folding in the inflammatory environment. Cytokines also regulated novel interactions between proinsulin and type 1 and type 2 diabetes genome-wide association studies candidate proteins not previously known to interact with proinsulin (eg, Ataxin-2). Finally, cytokines induced proinsulin interactions with a cluster of microtubule motor proteins and chemical destabilization of microtubules with Nocodazole exacerbated cytokine induced proinsulin secretion. Together, the data shed new light on mechanisms by which diabetes-associated cytokines dysregulate β-cell function. For the first time, we show that even short-term exposure to an inflammatory environment reshapes proinsulin interactions with critical chaperones and regulators of the secretory pathway.

184-OR: Antigen-Presenting Cells Can Generate Hybrid Peptides Identified in Type 1 Diabetes

Type 1 Diabetes (T1D) is an autoimmune disease characterized by selective loss of pancreatic β-cells. Aberrant reactivity to insulin and insulin precursor peptides has long been considered important in the pathogenesis of T1D. Recently, T cells recognizing hybrid insulin peptides (HIPs) have been described, first in a rodent model and later in human T1D. A HIP is a CD4 T-cell epitope that is formed by the post-translational fusion of two peptide fragments with at least one of the peptide fragments derived from insulin or a precursor. HIP formation can occur intra- or intermolecularly. The role of these HIPs in the pathogenesis of T1D remains unclear. Others have proposed that HIPs are formed in β-cells during crinophagy of secretory granules. As ultrastructural studies have demonstrated that β-cells can transfer secretory vesicles to local antigen presenting cells (APCs) , we hypothesized that APCs could similarly generate HIPs via protease mediated transpeptidation. To test this, we manufactured peptide-containing nanocarriers mimicking β-cell secretory vesicles to transfer selected peptides to APCs in both mouse and human in vitro systems utilizing donor APCs and CD4 TCR transgenic T cells or a CD4 transductant reporter cells respectively. Here, we demonstrate the subsets of APCs which are capable of intramolecular and intermolecular HIP formation. We further demonstrate that this formation can be blocked by proteosome inhibition. Our study identifies a novel mechanism for the generation of HIPs recognized in T1D. Disclosure S.A.Khokhar: None. M.Liu: None. J.L.Gaglia: Advisory Panel; Dompé, Regeneron Pharmaceuticals Inc., Consultant; Vertex Pharmaceuticals Incorporated, Research Support; Avotres Inc., Dompé, Janssen Research & Development, LLC, Provention Bio, Inc., Stock/Shareholder; Vertex Pharmaceuticals Incorporated. Funding National Institutes of Health 5P30DK036836JDRF 5-ECR-2016-186-A-N

245-LB: An Ankyrin-B–Mediated Protein Chaperone Mechanism Modulates Pancreatic ß-Cell Susceptibility to Proteotoxicity-Induced ER Stress and Apoptosis

Impaired insulin secretion contributes to the development and progression of diabetes. β-cells initially cope with the genetic, environmental, and diabetogenic stresses that diminish insulin secretion or the peripheral response to it by increasing insulin production. However, sustained increases in the insulin precursor proinsulin (PI) place additional demand on the secretory and protein quality control (PQC) machineries of β-cells, triggering endoplasmic reticulum (ER) and oxidative stress, mitochondrial dysfunction, and β-cell death. Thus, understanding the mechanisms that regulate β-cell responses to increased insulin secretory requirements is critical to prevent β-cell failure. We reported that the cytoskeletal scaffolding protein ankyrin-B (AnkB) regulates insulin secretion by maintaining normal levels of the inositol-1,4,5-triphosphate receptor (IP3R) at the ER and intracellular calcium in β-cells. IP3R levels and insulin secretion were impaired in knock-in mice carrying the p.R1788W AnkB (AnkB-RW) variant associated with type 2 diabetes (T2D) in Caucasians and Hispanics. Here, we show that selective loss of AnkB or expression of the T2D AnkB-RW variant lead to reduced insulin secretion and accumulation of PI and islet specific amyloid polypeptide in β-cells. These changes are accompanied by enhanced induction of the maladaptive unfolded protein response and apoptosis. Interestingly, the T2D AnkB-RW variant lies within the binding site for the protein chaperone Dnajb1 (Hsp40) . Our results indicate that the Dnajb1-Hspa1a complex, which recognizes and delivers protein clients to Hsp70 chaperones to promote proper folding, refolding, or degradation, is downregulated in β-cells from β-cell-specific AnkB knockout or AnkB-RW mice. Increased proteotoxicity and reduced insulin secretion were also observed upon knockdown of Dnajb1 in rat INS1 832/13 cells. Together, our findings suggest that AnkB and its integration into the Dnajb1-Hspa1a pathway modulates PQC and promotes β-cell survival under diabetogenic conditions. Disclosure J. Tzeng: None. H. Hohmeier: None. D. Lorenzo: None.

Evaluating Pulses and Modified Fed-batch Feeding of Methanol to Increase Expression Level of Human Insulin Precursor in Pichia Pastoris High-Density Cultivation

Production of sufficient insulin at a more affordable price is necessary. The increase in the number of people living with diabetes puts more burden on healthcare and the economy. P. pastoris is a promising host to produce human insulin precursors at a high yield in minimal medium and secretes low levels of endogenous protein impurities. Production of the precursor involves several parameters, including glycerol concentration, culture density, methanol concentration, and medium composition. This study evaluated the effect of those parameters on protein expression in the flask culture. Subsequently, fermentation in the bioreactor was carried on according to the information obtained from flask culture. Methanol feeding to induce protein expression was undertaken by pulses and fed-batch modes. The fed-batch method was modified from a standard technique by incorporating constant flow rates with variable feed concentrations. Cell density was determined based on optical density measurement at 600 nm and dry cell weight. Tricine SDS-PAGE and reversed-phase HPLC conducted protein analysis. The pulse feeding produced higher precursor concentrations at ~445 mg/L than modified fed-batch feeding at ~267 mg/L. However, the modified fed-batch feeding can be an alternative to producing human insulin precursors when a standard fed-batch feeding with variable flow rates and 100% (v/v) methanol feed is difficult to apply.

Insulin receptor activation by proinsulin preserves synapses and vision in retinitis pigmentosa

Synaptic loss, neuronal death, and circuit remodeling are common features of central nervous system neurodegenerative disorders. Retinitis pigmentosa (RP), the leading cause of inherited blindness, is a group of retinal dystrophies characterized by photoreceptor dysfunction and death. The insulin receptor, a key controller of metabolism, also regulates neuronal survival and synaptic formation, maintenance, and activity. Indeed, deficient insulin receptor signaling has been implicated in several brain neurodegenerative pathologies. We present evidence linking impaired insulin receptor signaling with RP. We describe a selective decrease in the levels of the insulin receptor and its downstream effector phospho-S6 in retinal horizontal cell terminals in the rd10 mouse model of RP, as well as aberrant synapses between rod photoreceptors and the postsynaptic terminals of horizontal and bipolar cells. A gene therapy strategy to induce sustained proinsulin, the insulin precursor, production restored retinal insulin receptor signaling, by increasing S6 phosphorylation, without peripheral metabolic consequences. Moreover, proinsulin preserved photoreceptor synaptic connectivity and prolonged visual function in electroretinogram and optomotor tests. These findings point to a disease-modifying role of insulin receptor and support the therapeutic potential of proinsulin in retinitis pigmentosa.

Proinsulinoma of the Pancreas. New Functioning Neuroendocrine Tumor. The Major Single-Center Study

Introduction: Proinsulinoma is an extremely rare neuroendocrine tumor of the pancreas which secretes an excessive amount of the insulin precursor. There are only case reports presented in the world literature about this neuroendocrine tumor.

Transformation and characterization of human insulin precursor gene in Pichiapastoris X-33

The case of diabetes increases significantly and has been projected to reach 592 million people in 2035. Consequently, the necessity of insulin will rise manifold and an efficient production system for insulin production is required to meet the market demands. The human insulin precursors that enzymatically converted to human insulin can be produced using Escherichia coli, Saccharomyces cerevisiae, or Pichia pastoris. In this study, Pichia pastoris is used for production human insulin precursor because the resulting recombinant protein can be folded accordingly and secreted to the external environment of the cell that simplifies the purification process. The study was initiated with the insertion of a synthetic gene of human insulin precursor into the pPICZaA to create recombinant pPICZaA-IP plasmid. The recombinant plasmid was transformed into Escherichia coli Top10 which then isolated and digested by the SacI enzyme. The linearize pPICZaA-IP plasmid was transfected into Pichia pastoris X-33 by electroporator. The result of transformation process, a total of 20 colonies of P pastoris X-33 were selected and inoculated in YPD agar medium containing Zeocin. The two colonies of P pastoris were characterized by PCR and sequencing showed that the recombinant pPICZaA-IP plasmid was successfully integrated into selected colonies of P pastoris.

Enhancement in Human Insulin Precursor Secretion by Pichia pastoris through Modification of Expression Conditions

Pichia pastoris is an alternative yeast expression system to produce heterologous proteins. It has excellent characteristics for an industrial cell factory, such as its ability to reach high cell densities, high secretory capacity, and a low level of native proteins. In our previous study, we introduced a synthetic insulin precursor (IP)-encoding gene constructed in a pD902 expression vector into P. pastoris. However, the P. pastoris recombinant strains expressed a little amount of IP protein. Here, we modified the expression conditions, including inoculum density, methanol concentration, methanol induction time, pH, and temperature, to obtain a higher amount of secreted IP than our previous result. Protein analysis for studying the five parameters was conducted by SDS-PAGE, and the protein amount was estimated by ImageJ applying lysozyme as standard. We successfully enhanced the IP expression by modifying expression conditions. The highest increased of up to 100 folds was achieved when methanol concentration for induction was arranged at 3% (v/v), and the initial cell density for methanol induction was set at an optical density at 600 nm (OD600) of approximately 10 compared to the standard procedure, where the expression was set at 0.5% (v/v) methanol induction and initial cell density at OD600 = 1.

UV-light-actuated in-situ preparation of paper@ZnCd quantum dots for paper-based enzymatic nanoreactors

Quantum dots (QDs) have been widely applied in the analytical field including sensitive fluorescent assays on paper-based devices. A facile in-situ synthesis of QDs after a simple reagent deposition on paper could enable a rapid low-cost fabrication of QD–modified paper with a variety of properties and purposes such as enzymatic nanoreactors. Herein, for the first time, fluorescent ZnCd QDs were prepared by in-situ synthesis in the paper matrix using thiol-containing precursors and irradiation by UV light. A successful creation of the immobilized ZnCd QDs on paper (paper@ZnCd QDs) was monitored by their intrinsic fluorescence while their peroxidase mimetic activity was evaluated by a catalytic reaction between H2O2 and a substrate (3,3′,5,5′-tetramethylbenzidine, TMB) producing a blue coloured product of charged transfer complex of TMB (oxTMB). From three thiol precursors investigated, the insulin precursor provided a greater activity than glutathione (GSH) and both considerably larger than BSA. Finally, a QDs precursor mixture deposition onto the paper matrix was evaluated by the peroxidase-mimetic activity, which was comparable with a reference method. The in-situ preparation of paper@ZnCd QDs is simple, rapid and ‘green’, with potential in biomedical applications primarily as fluorescence imaging agent and enzyme mimetic paper-based nanoreactors.

ELEMENTAL HOMEOSTASIS IN WISTAR RATS AS A MANIFESTATION OF ADAPTATION IN AN EXPERIMENT WITH METAL LOADING IN THE FORM OF MICRO- AND NANOPARTICLES

Relevance. In recent years, various methods have been widely used to determine the state of adaptation.One of the mechanisms providing adequate adaptation reactions and resistance of the organism to the action of hazardous environmental factors is an increase in the number of intra- and intersystem connections between the content of macronutrients and trace elements, as a means of reliable functioning of the organism.
 Objective. To reveal the typical stages of adaptation of the organism of rats in different conditions of the action of metals by studying the correlation dependences between the content of macronutrients and trace elements in various biological media and assessing the degree of adaptation of the elemental system.
 Materials and methods. Determination of the content of 14 chemical elements in whole blood and endocrine organs was performed using microwave mineralization of samples and was determined by the OES-ICP method on an Optima 2100 DV device. The assessment of their ratios and the analysis of correlations between the elements and the assessment of the degree of resistance of the organism by calculating the index - the degree of adaptation of the elemental system of the organism (A) according to the formula of R.M. Baevsky were carried out.
 Results. The degree of a, sti of experimental animals to the action of Al and Ag, Cr and Mn compounds was estimated according to the proposed by R. M. Baevsky et al. [4] the formula, taking into account the correlations between of macronutrients and trace elements in biological media (degree of adaptation - A). During intraperitoneal administration of metal salts, they were characterized by a stress stage for Al and Cr, the training stages corresponded to a change in the balance of elements under conditions of Mn salt administration. No significant violations of adaptation were found under the conditions of Ag action. Under the conditions of oral administration of metal salts, the degree of adaptation was higher for Ag and Al compounds, the stress stage was characterized by the effect of Mn and Cr salts on elemental homeostasis.
 Comparison of the action of NPs metal showed a greater adaptation to the action of NPs of Al and Ag oxides and worse to the action of NPs of Mn and Cr citrates. Sex differences in adaptation of animals to the action of metal salts were also revealed. Thus, in males exposed to Al, adaptation was characterized by a training stage, while in females the response to Ag action corresponded to the stress stage. The latter gives grounds to assert that the thyroid gland is more sensitive to Ag than in males. Correlation dependences between the content of metals and hormones in animals, exposed to compounds Ag, Al, Cr, Mn, showed a multidirectional effect of Ag and Al on the correlation between the content of metal and hormone I-TG If a direct correlation was found in the control between the content of metals in the blood and free T4 for animals of all groups, and in the experiment only under the condition of exposure to Al and Cr. For the hormone C-peptide (insulin precursor), a significant correlation was found. The bond was found only under the condition of exposure to Al and Mn.
 Conclusions. For the first time, an approach was applied to assessing the adaptation of an organism in terms of the balance of macronutrients and trace elements as an intermediate link in the adaptation process of the endocrine system in animal experiments. Comparison of the action of metal NPs showed a greater adaptation to the action of NPs of Al and Ag oxides and worse to the action of NPs of Mn and Cr citrates. Comparison of the effects of MC effect of metal nitrates and NPs of their oxides and hydrates according to the degree of adaptation of the elementary system indicates a higher toxicity of AgNO3, Al (NO3) 3, while the degree of adaptation under the action of NPs Mn-Ctr and Cr-Ct indicates their higher toxicity. Females were found to be more sensitive to Ag2O NPs than males, and the opposite effect was found under the conditions of Al2O3 NPs. The data obtained testify in favor of the existing ideas about the higher toxicity of metals in the form of nanoparticles compared to microparticles, that is, NPs of manganese and chromium citrates are more toxic.

25-OR: Role for TBC1D30 in Secretion of Mature Insulin

Genome-wide association studies (GWAS) have identified loci associated with the processing and secretion of mature insulin. Increased proinsulin (PI) secretion due to incomplete processing of insulin precursors has been linked to pancreatic beta-cell stress. At one PI GWAS locus, rs150781447-T is associated with increased PI secretion in Finns (p=5.5X10-11) and causes an amino acid (AA) change (R279C) at a conserved position in exon 4 of TBC1D30, a putative Rab GTPase Activating Protein (GAP), while no GWAS proxy variants (r2>0.8, 1000G Finns) are located in transcribed or enhancer regions. We hypothesized that TBC1D30 is involved in PI processing and that missense variant rs150781447, predicted to be damaging, has a negative impact on gene function. We used genome editing to generate four sets of TBC1D30 mutants in 832/13-INS1 cells. Compared to mock-edited control cells, apparent loss-of-function in frame insertion-deletion cells (106% expression) secreted 2.1-fold more PI (p=0.0001), and heterozygous truncation knockout cells (44% expression) secreted 1.9-fold more PI (p=0.005). Cells heterozygous for rs150781447, generated by prime editing, secreted 1.2-fold more PI (p=0.89), suggesting that the heterozygous variant has subtle or no effect on gene function. Unexpectedly, when exon 4 (42 AA) was deleted in frame, the cells secreted 3.2-fold less PI (p=0.02), consistent with a gain-of-function effect and suggesting that exon 4, part of the conserved Rab GAP protein domain, has a critical function. Preliminary insulin secretion data, while variable, demonstrate trends similar to proinsulin secretion, consistent with a stronger effect of TBC1D30 on processing than secretion. Together, these data demonstrate TBC1D30 as the likely target of the nearby PI GWAS locus and suggest that Rab GAP TBC1D30 plays a critical role in the processing of mature insulin. Disclosure V. A. Parsons: None. S. Vadlamudi: None. A. H. Moxley: None. K. L. Mohlke: None. Funding National Institutes of Health (1T32GM135128-01)

292-OR: Inflammatory Cytokines Rewire the Proinsulin Interaction Network in Human Islets

It has become increasingly appreciated that inflammatory cytokines promote islet dysfunction in both type 1 and type 2 diabetes. Aberrant secretion of the insulin precursor proinsulin is observed in patients with both types of diabetes and may contribute to proinsulin autoimmunity in T1D, yet the mechanisms responsible for proinsulin release remain obscure. To investigate the effects of inflammation on proinsulin trafficking, human islets were treated ex vivo with the diabetes associated cytokines, Interleukin-1-beta and Interferon-gamma, provoking a four-fold release of proinsulin. To identify proinsulin interacting proteins responsible for aberrant trafficking, intracellular proinsulin was affinity purified and its interactome defined by mass spectrometry. Cytokines altered the identify or magnitude of proinsulin’s engagement with binding partners at every stage of the canonical proinsulin biosynthetic pathway that we recently defined in normal human islets. Of particular relevance to potential avenues of proinsulin escape, cytokines promoted proinsulin interactions with: 1) antigen presentation loading complex components TAP2 and calnexin 2) stress granule assembly factors G3BP1, Ataxin-2 and IGF2BP2, that are also found in secreted exosomes, and 3) the immature insulin granule marker and T1D autoantigen ICA1/ICA169. Pathway analysis further revealed that cytokine treatment increased proinsulin binding, even if indirectly, to multiple KIF family microtubule motor proteins. Given that stress granules and immature granules are sensitive to microtubule dynamics we tested the role of microtubules in cytokine induced proinsulin release. Microtubule destabilization with Nocodazole significantly increased proinsulin release above that provoked by cytokines alone. Together, the data quantitatively map the cytokine reshaped proinsulin interactome and provide new insight into how an inflammatory environment can promote proinsulin escape from human islets. Disclosure D. T. Tran: Employee; Self; Encodia Inc. S. Loguercio: None. K. Lee: None. A. Pottekat: None. W. E. Balch: None. P. Arvan: None. R. J. Kaufman: None. P. Itkin-ansari: None. Funding JDRF (2-SRA-2015-47-M-R); National Institutes of Health (R24DK110973)