Insulin C-peptide Research Articles

Functional expression of recombinant insulins in Saccharomyces cerevisiae

BackgroundSince 1982, recombinant insulin has been used as a substitute for pancreatic insulin from animals. However, increasing demand in medical and food industries warrants the development of more efficient production methods. In this study, we aimed to develop a novel and efficient method for insulin production using a yeast secretion system.MethodsHere, insulin C-peptide was replaced with a hydrophilic fusion partner (HL18) containing an affinity tag for the hypersecretion and easy purification of proinsulin. The HL18 fusion partner was then removed by in vitro processing with the Kex2 endoprotease (Kex2p), and authentic insulin was recovered via affinity chromatography. To improve the insulin functions, molecular chaperones of the host strain were reinforced via the constitutive expression of HAC1.ResultsThe developed method was successfully applied for the expression of cow, pig, and chicken insulins in yeast. Moreover, biological activity of recombinant insulins was confirmed by growth stimulation of cell line.ConclusionsTherefore, replacement of the C-peptide of insulin with the HL18 fusion partner and use of Kex2p for in vitro processing of proinsulin guarantees the economic production of animal insulins in yeast.

7942 Evolution of Insulin, Insulin-like Growth Factor and Their Cognate Receptors in Vertebrates, Invertebrates and Viruses

Abstract Disclosure: M. Chrudinova: None. Y.D. Dogru: None. R. Huang: None. R. Reiners: None. P. De Meyts: None. J. DaCosta: None. E. Altindis: None. The insulin/insulin-like growth factor (IGF) signaling system is an evolutionarily conserved system and controls central processes including metabolism, cellular growth, proliferation, and differentiation in vertebrates. On the other hand, invertebrate insulin-like peptides (ILPs) regulate growth, stress responses, longevity and sex-related functions. In addition, we recently showed that six viruses possess viral insulin-like peptides (VILPs) that can bind to the receptors and stimulate post-receptor signaling. The evolution of these hormones and the origin of VILPs are not completely understood. Here, we collected vertebrate and invertebrate insulin, IGF and their cognate receptor sequences to analyze their evolutionary relations. We analyzed 177 vertebrate insulins, 119 vertebrate IGF-1s, 127 vertebrate IGF-2s, 197 invertebrate ILPs and 6 viral insulin/IGF-1 like peptides. We also analyzed 214 insulin receptor (IR), 160 IGF receptor (IGF1R) and 39 invertebrate receptor sequences to identify conservation and the evolutionary trajectory to reveal conserved motifs. Through phylogenetic analyses, we observed a separate branching of invertebrate ILPs and vertebrate insulins, IGF-1s and IGF-2s with notable exceptions including VILPs. IGF-1 and IGF-2 ligands were evolutionally better conserved compared to insulin ligands. Insulin C-peptide which is processed and cleaved was not well conserved compared to the functional A- and B-chain residues. In contrast, C-peptide of IGF-1 that plays an important role in receptor binding was very well conserved. IGF-1 C-domain showed higher variability in IGF1R binding residues compared to the A and B-domains, while residues important for binding of IGF-2 to IGF1R were well conserved. Insulin residues interacting with IR binding Site 1 were more conserved than those interacting with IR binding Site 2, while residues important for insulin dimerization and hexamerization residues are well conserved across species. We also showed that VILPs contain 14 amino acids that are conserved in insulin and IGFs, indicating their functional importance. Phylogenetic relationships of the receptor sequences suggest vertebrate IRs and IGF1Rs evolved from a common ancestor during early vertebrate evolution. Interestingly, receptor subunits revealed a closer relation of amphibian IR α-subunits to Mammalia IR α-subunits than Aves and Reptilians. The conservation rate of receptor residues essential for ligand-receptor interaction revealed that these residues are best conserved in the L1 domain for IR and IGF1R, with a higher rate of variation in other domains for both receptors. Taken together, our study provides a comprehensive analysis of insulin, IGFs, VILPs and their cognate receptors’ evolution, and opens new avenues to better understand the evolution, function, and structure relations of this complex signaling system. Presentation: 6/3/2024

Optimizing expression, purification, structural and functional assessments of a novel dimeric incretin (GLP-1cpGLP-1)

Glucagon-like peptide-1 (GLP-1) is an incretin hormone that reduces postprandial glycemic excursions by enhancing insulin secretion. In this study, a new dimeric GLP-1 analogue (GLP-1cpGLP-1) was designed by inserting human insulin C-peptide (CP) in the middle of a dimer of [Gly8] GLP-1 (7–36). Then, the dimeric incretin (GLP-1cpGLP-1) was ligated to human αB-crystallin (αB-Cry) to create a hybrid protein, abbreviated as αB-GLP-1cpGLP-1. The constructed gene was well expressed in the bacterial host system. After specific chemical release from the hybrid protein, the dimeric incretin was purified by size exclusion chromatography (SEC). Finally, the RP-HPLC analysis indicated a purity of >99 % for the dimeric incretin. The secondary structure assessments by various spectroscopic methods, and in silico analysis suggested that the dimeric incretin has α-helical rich structure. The dynamic light scattering (DLS) analysis indicates that our dimeric incretin forms large oligomeric structures. This incretin analogue significantly reduced blood glucose levels in both healthy and diabetic mice while effectively triggering insulin release. The size exclusion HPLC also indicates the interaction of the new incretin analogue with human serum albumin, the main carrier protein in the bloodstream. Consistent with the results obtained from the biological activity assessments, this significant interaction indicates its potential as a viable therapeutic agent with a long-lasting effect. The results of our research represent a significant breakthrough in the successful design of an active incretin dimer capable of effectively controlling blood sugar levels and inducing insulin secretion in the realm of diabetes treatment.

C-peptide and metabolic outcomes in trials of disease modifying therapy in new-onset type 1 diabetes: an individual participant meta-analysis

Metabolic outcomes in type 1 diabetes remain suboptimal. Disease modifying therapy to prevent β-cell loss presents an alternative treatment framework but the effect on metabolic outcomes is unclear. We, therefore, aimed to define the relationship between insulin C-peptide as a marker of β-cell function and metabolic outcomes in new-onset type 1 diabetes. 21 trials of disease-modifying interventions within 100 days of type 1 diabetes diagnosis comprising 1315 adults (ie, those 18 years and older) and 1396 children (ie, those younger than 18 years) were combined. Endpoints assessed were stimulated area under the curve C-peptide, HbA1c, insulin use, hypoglycaemic events, and composite scores (such as insulin dose adjusted A1c, total daily insulin, U/kg per day, and BETA-2 score). Positive studies were defined as those meeting their primary endpoint. Differences in outcomes between active and control groups were assessed using the Wilcoxon rank test. 6 months after treatment, a 24·8% greater C-peptide preservation in positive studies was associated with a 0·55% lower HbA1c (p<0·0001), with differences being detectable as early as 3 months. Cross-sectional analysis, combining positive and negative studies, was consistent with this proportionality: a 55% improvement in C-peptide preservation was associated with 0·64% lower HbA1c (p<0·0001). Higher initial C-peptide levels and greater preservation were associated with greater improvement in HbA1c. For HbA1c, IDAAC, and BETA-2 score, sample size predictions indicated that 2-3 times as many participants per group would be required to show a difference at 6 months as compared with C-peptide. Detecting a reduction in hypoglycaemia was affected by reporting methods. Interventions that preserve β-cell function are effective at improving metabolic outcomes in new-onset type 1 diabetes, confirming their potential as adjuncts to insulin. We have shown that improvements in HbA1c are directly proportional to the degree of C-peptide preservation, quantifying this relationship, and supporting the use of C-peptides as a surrogate endpoint in clinical trials. JDRF and Diabetes UK.

Insulin C-peptide secretion on-a-chip to measure the dynamics of secretion and metabolism from individual islets

First-phase glucose-stimulated insulin secretion is mechanistically linked to type 2 diabetes, yet the underlying metabolism is difficult to discern due to significant islet-to-islet variability. Here, we miniaturize a fluorescence anisotropy immunoassay onto a microfluidic device to measure C-peptide secretion from individual islets as a surrogate for insulin (InsC-chip). This method measures secretion from up to four islets at a time with ∼7s resolution while providing an optical window for real-time live-cell imaging. Using the InsC-chip, we reveal two glucose-dependent peaks of insulin secretion (i.e., a double peak) within the classically defined 1st phase (<10min). By combining real-time secretion and live-cell imaging, we show islets transition from glycolytic to oxidative phosphorylation (OxPhos)-driven metabolism at the nadir of the peaks. Overall, these data validate the InsC-chip to measure glucose-stimulated insulin secretion while revealing new dynamics in secretion defined by a shift in glucose metabolism.

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

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

Normative range of various serum hormonal parameters among Indian women of reproductive age: ICMR-PCOS task force study outcome.

The hormonal profile varies considerably with age, gender, ethnicity, diet or physiological state of an individual. Limited population-specific studies have studied the variations in hormonal parameters among apparently healthy women. We aimed to analyse the biological reference interval for various hormonal parameters in the reproductive-aged healthy Indian women. Out of 3877 participants that were clinically evaluated, 1441 subjects were subjected to laboratory investigations. All participants underwent a detailed clinical, biochemical and hormonal profiling. The hormone analysis was carried out at a single centre using a uniform methodology. Among the participants evaluated for biochemical and hormonal parameters, subjects that presented any abnormal profile or had incomplete investigations (n=593) were excluded for further analysis. The mean age (±SD) of the subjects retained in the final analysis (n=848) was 29.9 (±6.3) years. In the present study, the biological reference interval (2.5th-97.5th centile) observed were: serum T4: μg/dL (5.23-12.31), TSH: μg/mL (0.52-4.16) and serum prolactin: ng/mL (5.13-37.35), LH: mIU/mL (2.75-20.68), FSH: mIU/mL 2.59-15.12), serum total testosterone: ng/mL (0.06-0.68), fasting insulin: mIU/mL (1.92-39.72), morning cortisol: μg/dL (4.71-19.64), DHEAS:μg/dL (50.61-342.6) and SHBG: nmol/L (21.37-117.54). Unlike T4, TSH, LH, and E2, the biological reference interval for prolactin, FSH, testosterone, C-peptide insulin and DHEAS varied when the subjects were stratified by age (p<0.05). The comparative analysis showed marginal differences in the normative ranges for the hormones analysed among different populations. Our first large composite data on hormonal measures will benefit future endeavours to define biological reference intervals in reproductive-aged Indian women. The study was financially supported by the grant-in-aid from ICMR vide file No:5/7/13337/2015-RBMH.

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

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

Sex-specific responses in glucose-insulin homeostasis and lipoprotein-lipid components after high-dose supplementation with marine n-3 PUFAs in abdominal obesity: a randomized double-blind crossover study.

Clinical studies on effects of marine-derived omega-3 (n-3) polyunsaturated fatty acids (PUFAs), mainly eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), and the plant-derived omega-6 (n-6) PUFA linoleic acid (LA) on lipoprotein-lipid components and glucose-insulin homeostasis have shown conflicting results, which may partly be explained by differential responses in females and males. However, we have lacked data on sexual dimorphism in the response of cardiometabolic risk markers following increased consumption of n-3 or n-6 PUFAs. To explore sex-specific responses after n-3 (EPA + DHA) or n-6 (LA) PUFA supplementation on circulating lipoprotein subfractions, standard lipids, apolipoproteins, fatty acids in red blood cell membranes, and markers of glycemic control/insulin sensitivity among people with abdominal obesity. This was a randomized double-blind crossover study with two 7-week intervention periods separated by a 9-week washout phase. Females (n = 16) were supplemented with 3 g/d of EPA + DHA (fish oil) or 15 g/d of LA (safflower oil), while males (n = 23) received a dose of 4 g/d of EPA + DHA or 20 g/d of LA. In fasting blood samples, we measured lipoprotein particle subclasses, standard lipids, apolipoproteins, fatty acid profiles, and markers of glycemic control/insulin sensitivity. The between-sex difference in relative change scores was significant after n-3 for total high-density lipoproteins (females/males: -11%*/-3.3%, p = 0.036; *: significant within-sex change), high-density lipoprotein particle size (+2.1%*/-0.1%, p = 0.045), and arachidonic acid (-8.3%*/-12%*, p = 0.012), and after n-6 for total (+37%*/+2.1%, p = 0.041) and small very-low-density lipoproteins (+97%*/+14%, p = 0.021), and lipoprotein (a) (-16%*/+0.1%, p = 0.028). Circulating markers of glucose-insulin homeostasis differed significantly after n-3 for glucose (females/males: -2.1%/+3.9%*, p = 0.029), insulin (-31%*/+16%, p < 0.001), insulin C-peptide (-12%*/+13%*, p = 0.001), homeostasis model assessment of insulin resistance index 2 (-12%*/+14%*, p = 0.001) and insulin sensitivity index 2 (+14%*/-12%*, p = 0.001), and quantitative insulin sensitivity check index (+4.9%*/-3.4%*, p < 0.001). We found sex-specific responses after high-dose n-3 (but not n-6) supplementation in circulating markers of glycemic control/insulin sensitivity, which improved in females but worsened in males. This may partly be related to the sex differences we observed in several components of the lipoprotein-lipid profile following the n-3 intervention. https://clinicaltrials.gov/, identifier [NCT02647333].

Insulin B-chain hybrid peptides are agonists for T cells reactive to insulin B:9-23 in autoimmune diabetes.

Insulin is considered to be a key antigenic target of T cells in Type 1 Diabetes (T1D) and autoimmune diabetes in the NOD mouse with particular focus on the B-chain amino acid sequence B:9-23 as the primary epitope. Our lab previously discovered that hybrid insulin peptides (HIPs), comprised of insulin C-peptide fragments fused to other β-cell granule peptides, are ligands for several pathogenic CD4 T cell clones derived from NOD mice and for autoreactive CD4 T cells from T1D patients. A subset of CD4 T cell clones from our panel react to insulin and B:9-23 but only at high concentrations of antigen. We hypothesized that HIPs might also be formed from insulin B-chain sequences covalently bound to other endogenously cleaved ß-cell proteins. We report here on the identification of a B-chain HIP, termed the 6.3HIP, containing a fragment of B:9-23 joined to an endogenously processed peptide of ProSAAS, as a strong neo-epitope for the insulin-reactive CD4 T cell clone BDC-6.3. Using an I-Ag7 tetramer loaded with the 6.3HIP, we demonstrate that T cells reactive to this B-chain HIP can be readily detected in NOD mouse islet infiltrates. This work suggests that some portion of autoreactive T cells stimulated by insulin B:9-23 may be responding to B-chain HIPs as peptide ligands.

Abstract CT518: First-in-human dose-escalation study of the phosphatidylinositol 3-kinase α-selective inhibitor HS-10352 in advanced breast cancer

Abstract Background: PIK3CA mutation is particularly prevalent in hormone receptor-positive (HR+), human epidermal growth factor receptor 2-negative (HER2-) breast cancer. HS-10352 is a highly potent and selective small molecule inhibitor of phosphoinositide 3-kinase α. Preclinical studies have indicated the favorable safety and antitumor activity of HS-10352 in PIK3CA-mutated tumor models. Methods: A phase 1, open-label, dose-escalation study was conducted to assess the safety, tolerability, pharmacokinetics (PK) and preliminary anti-tumor activity, in patients (pts) with advanced HR+HER2- breast cancer for whom standard therapy was ineffective or unavailable. The rolling six dose-escalation scheme was used to determine the maximum tolerated dose (MTD). Tumor tissues were collected for retrospectively central determination of PIK3CA mutation status. Results: Eighteen pts were treated with HS-10352 orally once daily (2 mg n=3; 4 mg n=3; 6 mg n=6; 8 mg n=6). All patients were HR+HER2- female breast cancer with visceral metastases after a median number of 3 prior systemic therapies for advanced disease. The median treatment duration was 111.5 days (range: 13~388). Dose limiting toxicities occurred in 2 pts at 8 mg (2 Grade 3 hyperglycemia). The MTD was established as 6 mg. According to the CTCAE 5.0, the most common (≥ 30%) treatment-related adverse events (TRAEs) were hyperglycemia (88.9%, n=16), weight decreased (61.1%, n=11), insulin C-peptide increased and diarrhea (33.3% each, n=6). Grade 3-4 TRAEs were hyperglycemia (8 mg, n=2), weight decreased (6 mg, n=2), fatigue (8 mg, n=1), vision blurred (8 mg, n=1), hyperkalemia (8 mg, n=1) and hypocalcemia (8 mg, n=1). Hyperglycemia was generally manageable with oral anti-hyperglycemic medications (13/16). Treatment-related rash were mild (22.2%, grade 1: n=2; grade 2: n=2). The overall response rate (ORR) and disease control rate (DCR) for all patients (n=18) were 27.8% and 55.6% respectively. For 6 pts with PIK3CA mutation, ORR was 50.0% (confirmed partial response [PR] in 3 pts at 6 mg) and DCR was 100.0% (6 mg, n=4; 8 mg, n=2), and the 9-month progression-free survival (PFS) rate was 66.7% (95% CI: 28.9%~100.0%). Of 4 pts with PIK3CA mutation at potentially recommended phase 2 dose of 6 mg, three pts (75.0%) achieved confirmed PR and 1 patient (25.0%) had stable disease (PFS ≥ 7.4m). Plasma exposure of HS-10352 increased proportionally following single dose. The mean plasma half-life of HS-10352 was 11~15 hours. Conclusions: HS-10352 showed a manageable safety profile and favorable PK properties. The promising antitumor activity was observed in patients with HR+HER2- advanced breast cancer harboring PIK3CA mutation. Clinical trial information: NCT04631835. Citation Format: Quchang Ouyang, Ying Wang, Jian Zhang, Qiong Wu, Changan Sun, Hongying Wei, Xiaoling Qian, Xichun Hu. First-in-human dose-escalation study of the phosphatidylinositol 3-kinase α-selective inhibitor HS-10352 in advanced breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT518.

Obesity in adolescents associated with vascular aging - a study using ultra-high-resolution ultrasound.

BackgroundObesity in adolescents is increasing worldwide and associated with an elevated cardiovascular risk later in life. In a group-comparative study, we investigated the association between adiposity in adolescents and signs of vascular aging and inflammation.MethodsThirty-nine adolescents (10–18 years old), 19 with obesity and 20 with normal weight, were enrolled. The intima thickness and intima/media thickness ratio (I/M) were assessed using high-resolution ultrasound in the common carotid artery (center frequency 22 MHz) and the distal radial artery (RA; 50 MHz). Increased intima and high I/M are signs of vascular aging. Body characteristics, high-sensitivity C-reactive protein (hs-CRP), plasma lipids, and glycemic parameters were measured.ResultsAdolescents with obesity, compared to normal-weight peers, had elevated plasma lipid, insulin c-peptide, and hs-CRP levels, the latter increasing exponentially with increasing adiposity. Obese adolescents had a thicker RA intima layer [0.005 mm; 95% confidence intervals (0.000, 0.009); P = 0.043] and a higher RA I/M [0.10; (0.040, 0.147); P < 0.0007]. Group differences for the RA I/M remained significant after adjustment for age, sex, fasting plasma insulin, and body mass index, both separately and together (P = 0.032). The RA I/M was correlated with hs-CRP, and both were correlated with the analyzed cardiovascular risk factors. Receiver operating curve c-values for RA I/M (0.86) and hs-CRP (0.90) strongly indicated correct placement in the obese or non-obese group.ConclusionsAdolescents with obesity had significantly more extensive vascular aging in the muscular RA, than normal-weight peers. The findings support an inflammatory link between obesity and vascular aging in adolescents.

188-OR: Metabolic Blockade of Glycolysis Can Delay the Adoptive Transfer of Type 1 Diabetes

Type 1 diabetes (T1D) is a result of insulin-secreting pancreatic β-cell destruction by autoreactive CD4 and CD8 T cells. There is no cure for T1D, but determining the role of immunometabolism in promoting autoreactive CD4+ T cell effector responses may provide a novel target in delaying T1D. Systemically inhibiting glycolysis with 2-deoxyglucose (2-DG) can decrease autoreactive CD8 T cell islet infiltration. However, the effects of 2-DG on CD4 T cell diabetogenicity is unknown. Proinflammatory Th1 cells mediate β-cell destruction prefer glycolysis for maintaining effector responses, while immunosuppressive Treg cells prefer oxidative phosphorylation. We hypothesize that metabolic inhibition of glycolysis will diminish proinflammatory Th1 cell responses and delay β-cell destruction in T1D. Utilizing 2-DG, we performed an adoptive transfer of diabetogenic CD4+ T cells from NOD.BDC-2.5 or NOD.BDC-6.9 mice into NOD.scid mice. Recipient mice were systemically treated with 30 mM 2-DG in the drinking water beginning 4 days prior to adoptive transfer and monitored for hyperglycemia for 80 days post-transfer. NOD.scid mice treated with 2-DG had a significant delay in diabetes onset when transferred with either BDC-2.5 (n=14, p=0.0284) or BDC-6.9 (n=17, p=0.0249) CD4+ T cells compared to vehicle-treated groups. To define the effects of 2-DG on autoreactive CD4 T cell effector responses, NOD.BDC-6.9 splenocytes were in vitro stimulated with their cognate hybrid insulin peptide (HIP; insulin C-peptide and islet amyloid polypeptide) with or without 2-DG. Flow cytometry analysis revealed a decrease in T cell proliferation, T cell activation, and Th1 lineage commitment as shown by Ki67, CD25, and T-bet expression, respectively. Effector responses were also dampened as IFN-γ synthesis was decreased in our 2-DG treatment group compared to HIP only controls. Future studies will investigate the effects of 2-DG on CD4 T cell chemokine secretion, trafficking into the islet, and cellular metabolic profiles. Disclosure M.D.Chavez: None. R.Mcdowell: None. H.M.Tse: None.

112-OR: Metabolic Outcomes One Year after Total Pancreatectomy with Islet Autotransplantation after Early Pump Initiation

Background: We previously published that insulin pump initiation immediately after IV insulin therapy was associated with improved post-surgical glycemic outcomes compared to multiple daily injections (MDI) in pediatric patients following total pancreatectomy with islet autotransplantation (TPIAT) . We investigated metabolic outcomes of this population at 1 year post-TPIAT to assess if the improved outcomes in the early pump group were sustained over time. Methods: We retrospectively reviewed 40 patients post-TPIAT previously studied at days post-surgery (14 used MDI and 26 used pump therapy immediately post-ICU, all discharged on pump therapy) . Data analyzed included: demographics, islet equivalents per kilogram (IEQ/kg) transplanted, exogenous insulin use, and baseline vs. 1 year (via mixed meal testing) HgA1C, fasting glucose, insulinogenic index and area under the curve (AUC) for insulin and c-peptide. Results: More patients were off insulin at 1 year in the early pump group compared to the MDI group (43% vs. 14%, p=0.08) . Of all patients off insulin, 100% of the early pump users weaned off by 6 months post-TPIAT compared to 30% of the MDI users. Two known variables associated with favorable insulin outcomes, lower age and higher IEQ/kg were not significantly different between groups. Fasting glucose was lower in the early pump group compared to the MDI group (median 97.5 vs. 122 mg/dL, p=0.01) while AUC c-peptide was greater in early pump users at 1 year post-TPIAT but did not reach significance (median 65.3 vs. 49.5 ng/mL x minutes, p=0.07) . Other metabolic outcomes did not differ between groups. Conclusion: Despite lower median age and higher IEQ/kg in the MDI group, the early pump group had a lower fasting glucose. Though not significant, the early pump group had a higher proportion off insulin and greater AUC c-peptide secretion. This study was limited by sample size. Early pump use may have long term benefits in post-TPIAT endogenous insulin secretion. Disclosure S.E.Tellez: None. L.Hornung: None. J.D.Courter: None. M.Abu-el-haija: None. J.D.Nathan: None. S.Lawson: None. D.A.Elder: None.

Maternal carbohydrate intake during pregnancy is associated with child peripubertal markers of metabolic health but not adiposity.

To examine the associations of trimester-specific maternal prenatal carbohydrate (CHO) intake with offspring adiposity and metabolic health during peripuberty. Prospective cohort study in which maternal dietary intake was collected via validated FFQ during each trimester. Offspring adiposity and metabolic biomarkers were evaluated at age 8-14 years. We used multivariable linear regression to examine associations between total energy-adjusted maternal CHO intake and offspring BMI z-score, skinfold thickness and metabolic syndrome risk z-score calculated as the average of waist circumference, fasting glucose, fasting C-peptide, TAG:HDL and systolic blood pressure + diastolic blood pressure/2. Mexico City, Mexico. 237 mother-child pairs in the Early Life Exposure in Mexico to Environmental Toxicants cohort. We found non-linear associations of maternal CHO intake during pregnancy with offspring metabolic health during peripuberty. After adjusting for maternal age, and child age, sex and pubertal status, children whose mothers were in the fourth v. first quartile of total CHO intake during the third trimester had 0·42 (95 % CI -0·01, 0·08) ng/ml lower C-peptide and 0·10 (95 % CI -0·02, 0·22) units lower C-peptide insulin resistance (CP-IR). We found similar magnitude and direction of association with respect to net CHO intake during the first trimester and offspring C-peptide and CP-IR. Maternal CHO intake during pregnancy was not associated with offspring adiposity. In this study of mother-child pairs in Mexico City, children born to women in the highest quartile of CHO intake during pregnancy had lowest C-peptide and CP-IR during peripuberty. Additional research is warranted to replicate and identify mechanisms.

Short-Term SGLT2 Inhibitor Administration Does Not Alter Systemic Insulin Clearance in Type 2 Diabetes.

Background: Decreased insulin clearance could be a relatively upstream abnormality in obesity, metabolic syndrome, and nonalcoholic fatty liver disease. Previous studies have shown that sodium-glucose cotransporter 2 inhibitor (SGLT2i) increases insulin–C-peptide ratio, a marker of insulin clearance, and improves metabolic parameters. We evaluated the effects of the SGLT2i tofogliflozin on metabolic clearance rate of insulin (MCRI) with a hyperinsulinemic euglycemic clamp study, the gold standard for measuring systemic insulin clearance. Methods: Study participants were 12 Japanese men with type 2 diabetes. We evaluated MCRI and tissue-specific insulin sensitivity with a hyperinsulinemic euglycemic clamp (insulin infusion rate, 40 mU/m2·min) before and immediately after a single dose (n = 12) and 8 weeks (n = 9) of tofogliflozin. We also measured ectopic fat in muscle and liver and the abdominal fat area using 1H-magnetic resonance spectroscopy and magnetic resonance imaging, respectively, before and after 8 weeks of tofogliflozin. Results: MCRI did not change after a single dose of tofogliflozin (594.7 ± 67.7 mL/min·m2 and 608.3 ± 90.9 mL/min·m2, p = 0.61) or after 8 weeks (582.5 ± 67.3 mL/min·m2 and 602.3 ± 67.0 mL/min·m2, p = 0.41). The 8-week treatment significantly improved glycated hemoglobin and decreased body weight (1.7%) and the subcutaneous fat area (6.4%), whereas insulin sensitivity and ectopic fat in muscle and liver did not change significantly. Conclusions: MCRI did not change after a single dose or 8 weeks of tofogliflozin. Increased MCRI does not precede a decrease in body fat or improved glycemic control.

A TCR-like antibody against a proinsulin-containing fusion peptide ameliorates type 1 diabetes in NOD mice

Type 1 diabetes (T1D) is an autoimmune disease caused by destruction of insulin-producing β cells. The response of autoreactive T cells to β cell antigens plays a central role in the development of T1D. Recently, fusion peptides composed by insulin C-peptide fragments and other proteins were reported as β cell target antigens for diabetogenic CD4+ T cells in non-obese diabetic (NOD) mice. In this study, we generated a T cell-receptor (TCR)-like monoclonal antibody (mAb) against a fusion peptide bound to major histocompatibility complex (MHC) class II component to elucidate the function of the fusion peptides in T1D. In addition, we developed a novel NFAT-GFP TCR reporter system to evaluate the TCR-like mAb. The NFAT-GFP reporter T cells expressing the diabetogenic TCR were specifically activated by the fusion peptide presented on the MHC class II molecules. By using the NFAT-GFP reporter T cells, we showed that the TCR-like mAb blocks the diabetogenic T cell response against the fusion peptide presented on the MHC class II molecules. Furthermore, the development of T1D was ameliorated when pre-diabetic NOD mice were treated with this mAb. These findings suggest that NFAT-GFP reporter T cells are useful to assess the function of specific TCR and the recognition of fusion peptides by T cells is crucial for the pathogenesis of T1D.

Circulating levels of selected adipokines in women with gestational diabetes and type 2 diabetes.

Adiponectin, adipocyte-fatty acid binding protein (A-FABP), and Wnt1 inducible signaling pathway protein-1 (WISP-1) are adipokines closely associated with insulin resistance. The aim of the study was to compare their levels in women with gestational diabetes (GDM), type 2 diabetes mellitus (T2DM) and healthy controls and determine their relation to metabolic parameters. Women with GDM, T2DM and healthy women were included in this cross-sectional study. In addition to adipokines, anthropometric, lipid parameters, markers of insulin resistance and glucose control were assessed in all participants. Compared to healthy controls (n = 35) significantly lower levels of adiponectin were detected in women with GDM (n = 50), whereas in women with T2DM (n = 50) higher levels of A-FABP and WISP-1 and lower levels of adiponectin were found. Women with T2DM had also lower levels of adiponectin and higher levels of A-FABP compared to women with GDM. A-FABP and adiponectin were independently associated with levels of triglycerides, HDL-cholesterol and C-peptide insulin resistance index. WISP-1 correlated only with waist circumference. Adverse adipokines production reflecting dysfunctional fat tissue is less presented in women with GDM than in women with T2DM, but more expressed compared to healthy women.

364-OR: Igls Classification for Islet Autotransplantation

Background: The Igls criteria for assessing islet function after allotransplantation cannot be directly applied to islet autotransplantation (IAT) patients due to differences in baseline characteristics of IAT patients (insulin independent and C-peptide positive). Methods: We tested modified criteria (Auto-Igls) for assessing islet function in a large cohort of total pancreatectomy-IAT (TPIAT) patients from 2010-2018 (n=379). Metabolic outcomes post-IAT were assessed (Table 1). We assigned an Auto-Igls class to each patient as able, and evaluated the utility, validity, and perioperative predictors of Auto-Igls at 1 year post-IAT. We associated Auto-Igls with independent measures of islet graft function where available: continuous glucose monitoring (CGM) data or acute c-peptide response to glucose (ACRglu) from intravenous glucose tolerance tests. Results: Auto-Igls class was assigned to 264 patients (69%). Of the 115 patients who could not be classified, most (74%) were missing exact insulin u/kg/day. The only significant predictor of Auto-Igls class was the islet mass transplanted (p&amp;lt;0.0001). Higher percent time in range (70-140 mg/dL) on CGM and higher ACRglu were associated with a better Auto-Igls class (p=0.02 and p&amp;lt;0.0001, respectively). Discussion: The modified Igls criteria for IAT permit a simple, comprehensive assessment of metabolic outcomes after TPIAT, and is associated with other measures of islet function. Disclosure K.R. McEachron: None. Y. Yang: None. T.L. Pruett: None. M. Bellin: Research Support; Self; Dexcom, Inc., Viacyte, Inc. Other Relationship; Self; Insulet Corporation. Funding National Institutes of Health (T32DK108733, R01DK109124)

962-P: Impacts of Different Hypoglycemic Agents on Adipokines and Inflammatory Markers in Newly Diagnosed T2D Patients

Background: Adipokines and inflammatory markers contribute to the pathogenesis of T2D. This study investigated the impacts of T2D and different hypoglycemic agents on the important adipokines and inflammatory markers in newly diagnosed T2D patients. Methods: The clinical parameters, leptin, adiponectin, resistin, fibroblast growth factor21, betatrophin, IL-1β and IFNγ were assessed in 41 normal-glucose controls and 416 newly diagnosed T2D patients. Then the patients were randomly assigned to receive exenatide, insulin or pioglitazone, with the above adipokines and inflammatory factors evaluated at 48 weeks. Results: As shown in Table 1, compared with the controls, T2D patients had significantly higher IL-1β, lower leptin, adiponectin and IFNγ levels. Among the 416 patients, 342 patients completed the 48-week study. The HbA1c change (ΔHbA1c) were similar among groups. Exenatide significantly decreased the BMI and IL-1β, while increased IFNγ and adiponectin. Insulin markedly increased IL-1β and IFNγ. And pioglitazone only increased adiponectin. Multiple linear regression showed that Δleptin and Δadiponectin were associated with ΔBMI, but not with ΔHbA1c, fasting insulin or fasting C-peptide. Conclusions: Exenatide has better impacts on improving IL-1β, IFNγ, and adiponectin levels than insulin and pioglitazone in T2D patients, which may probably due to decreasing BMI. Disclosure H. Deng: None. X. Yang: None. W. Xu: None. J. Lv: None. L. Zeng: None. B. Yao: None. J. Weng: None. Funding National Natural Science Foundation of China (81770821 to W.X.); National Key Research and Development Program of China (2016YFC1304801); Eli Lilly and Company