Beta Cell Stress Research Articles

Effect of fenofibrate on residual beta cell function in adults and adolescents with newly diagnosed type 1 diabetes: a randomised clinical trial.

Fenofibrate, a peroxisome proliferator-activated receptor alpha agonist, shows some promise in alleviating beta cell stress and preserving beta cell function in preclinical studies of type 1 diabetes. The aim of this phase 2, placebo-controlled, double-blinded, randomised clinical trial was to investigate the efficacy and safety of fenofibrate in adults and adolescents with newly diagnosed type 1 diabetes. We enrolled 58 individuals (aged 16 to 40 years old) with newly diagnosed type 1 diabetes and randomised them to daily oral treatment with fenofibrate 160 mg or placebo for 52 weeks (in a block design with a block size of 4, assigned in a 1:1 ratio). Our primary outcome was change in beta cell function after 52 weeks of treatment, assessed by AUC for C-peptide levels following a 2h mixed-meal tolerance test. Secondary outcomes included glycaemic control (assessed by HbA1c and continuous glucose monitoring), daily insulin use, and proinsulin/C-peptide (PI/C) ratio as a marker of beta cell stress. We assessed outcome measures before and after 4, 12, 26 and 52 weeks of treatment. Blinding was maintained for participants, their healthcare providers and all staff involved in handling outcome samples and assessment. The statistical analyses for the primary outcome included 56 participants (n=27 in the fenofibrate group, after two withdrawals, and n=29 in the placebo group). We found no significant differences between the groups in either 2h C-peptide levels (mean difference of 0.08 nmol/l [95% CI -0.05, 0.23]), insulin use or glycaemic control after 52 weeks of treatment. On the contrary, the fenofibrate group showed a higher PI/C ratio at week 52 compared with placebo (mean difference of 0.024 [95% CI 0.000, 0.048], p<0.05). Blood lipidome analysis revealed that fenofibrate repressed pathways involved in sphingolipid metabolism and signalling at week 52 compared with placebo. The 52 week intervention evoked few adverse events and no serious adverse events. Follow-up in vitro experiments in human pancreatic islets demonstrated a stress-inducing effect of fenofibrate. Contrary to the beneficial effects of fenofibrate found in preclinical studies, this longitudinal, randomised, placebo-controlled trial does not support the use of fenofibrate for preserving beta cell function in individuals with newly diagnosed type 1 diabetes. EudraCT number: 2019-004434-41 FUNDING: This study was funded by the Sehested Hansens Foundation.

Abstract PR-05: Endocrine beta-cell stress promotes pancreatic ductal adenocarcinoma through endocrine-exocrine cell crosstalk

Abstract For a long time, the pancreas was thought to have separate cellular compartments that functioned distinctly from one another. The endocrine pancreas (islets of Langerhans) regulates glucose homeostasis, while the exocrine pancreas (acini and ducts) produces and secretes digestive enzymes. However, it has recently become clear that the endocrine and exocrine compartments communicate with one another, and dysfunction in one leads to dysfunction in the other, resulting in diabetes or pancreatitis. However, whether and how the endocrine pancreas drives the development of pancreatic ductal adenocarcinoma (PDAC), an exocrine tumor, remains unresolved. Strikingly, we found that genetic ablation of insulin-producing islet beta (β) cells (Akita) in a faithful Kras/Trp53-driven PDAC model (KPC: Kras LSL-G12D /+; Trp 53172 /+; Pdx1-Cre) suppressed PDAC progression. Conversely, obesity-induced β cell hormone dysregulation promoted Kras-driven PDAC development. Single-cell RNA sequencing (scRNA-seq) analysis of wild-type and obese mice (high-fat diet-fed and leptin-deficient (Lep ob/ob )) revealed increased expression of the peptide hormone cholecystokinin (CCK) in a subset of β cells concordant with increasing obesity, and transgenic β cell overexpression of CCK was sufficient to promote exocrine tumorigenesis in KC mice. Combined in silico (pseudotime (TrajectoryNET) and archetypal (AANet) analysis) and experimental (CreER) lineage tracing demonstrated that CCK-expressing β cells originated from a pre-existing immature β cell population (virgin β cells). Grainger causality analysis of transcriptional networks uncovered a stress-induced JNK-cJun pathway that promotes CCK expression β cells, which we confirmed using JNK inhibitors in β cell models. Together, our findings identify cellular and molecular mechanisms of β cell adaptation to obesity that contribute to obesity-driven pancreatic cancer. Furthermore, we define a critical role for endocrine-exocrine signaling in PDAC progression and stress-induced β cell pathways which could be leveraged to target the endocrine pancreas to subvert exocrine tumorigenesis. Citation Format: Cathy Garcia, Aarthi Venkat, Daniel McQuaid, Sherry Agabiti, Alex Tong, Rebecca Cardone, Richard Kibbey, Smita Krishnaswamy, Mandar Muzumdar. Endocrine beta-cell stress promotes pancreatic ductal adenocarcinoma through endocrine-exocrine cell crosstalk [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr PR-05.

Immune checkpoints and pancreatic beta cell dysfunction in HIV.

We explored the impact of immune dysregulation on pancreatic beta cell injury in HIV patients. Analyzing 105 participant samples, we observed lower IL-21 levels and elevated immune checkpoint levels (e.g. PD-1, CD27+, CD40+) in untreated HIV patients. Notably, soluble TIM-3 correlated positively with improved beta cell function and inversely with beta cell stress, suggesting its potential role in beta cell protection in untreated HIV.

Extracellular vesicles derived from stressed beta cells mediate monocyte activation and contribute to islet inflammation.

Beta cell destruction in type 1 diabetes (T1D) results from the combined effect of inflammation and recurrent autoimmunity. In recent years, the role played by beta cells in the development of T1D has evolved from passive victims of the immune system to active contributors in their own destruction. We and others have demonstrated that perturbations in the islet microenvironment promote endoplasmic reticulum (ER) stress in beta cells, leading to enhanced immunogenicity. Among the underlying mechanisms, secretion of extracellular vesicles (EVs) by beta cells has been suggested to mediate the crosstalk with the immune cell compartment. To study the role of cellular stress in the early events of T1D development, we generated a novel cellular model for constitutive ER stress by modulating the expression of HSPA5, which encodes BiP/GRP78, in EndoC-βH1 cells. To investigate the role of EVs in the interaction between beta cells and the immune system, we characterized the EV miRNA cargo and evaluated their effect on innate immune cells. Analysis of the transcriptome showed that HSPA5 knockdown resulted in the upregulation of signaling pathways involved in the unfolded protein response (UPR) and changes the miRNA content of EVs, including reduced levels of miRNAs involved in IL-1β signaling. Treatment of primary human monocytes with EVs from stressed beta cells resulted in increased surface expression of CD11b, HLA-DR, CD40 and CD86 and upregulation of IL-1β and IL-6. These findings indicate that the content of EVs derived from stressed beta cells can be a mediator of islet inflammation.

A secondary analysis of indices of hepatic and beta cell function following 12 weeks of carbohydrate and energy restriction vs. free-living control in adults with type 2 diabetes

BackgroundSubstantial weight loss in people living with type 2 diabetes (T2D) can reduce the need for glucose-lowering medications while concurrently lowering glycemia below the diagnostic threshold for the disease. Furthermore, weight-loss interventions have also been demonstrated to improve aspects of underlying T2D pathophysiology related to ectopic fat in the liver and pancreatic beta-cell function. As such, the purpose of this secondary analysis was to explore the extent to which a low-carbohydrate and energy-restricted (LCER) diet intervention improves markers of beta-cell stress/function, liver fat accumulation, and metabolic related liver function in people with type 2 diabetes.MethodsWe conducted secondary analyses of blood samples from a larger pragmatic community-based parallel-group randomized controlled trial involving a 12-week pharmacist implemented LCER diet (Pharm-TCR: <50 g carbohydrates; ~850–1100 kcal/day; n = 20) versus treatment-as-usual (TAU; n = 16). Participants were people with T2D, using ≥ 1 glucose-lowering medication, and a body mass index of ≥ 30 kg/m2. Main outcomes were C-peptide to proinsulin ratio, circulating microRNA 375 (miR375), homeostatic model assessment (HOMA) beta-cell function (B), fatty liver index (FLI), hepatic steatosis index (HSI), HOMA insulin resistance (IR), and circulating fetuin-A and fibroblast growth factor 21 (FGF21). Data were analysed using linear regression with baseline as a covariate.ResultsThere was no observed change in miR375 (p = 0.42), C-peptide to proinsulin ratio (p = 0.17) or HOMA B (p = 0.15). FLI and HSI were reduced by -25.1 (p < 0.0001) and − 4.9 (p < 0.0001), respectively. HOMA IR was reduced by -46.5% (p = 0.011). FGF21 was reduced by -161.2pg/mL (p = 0.035) with a similar tendency found for fetuin-A (mean difference: -16.7ng/mL; p = 0.11). These improvements in markers of hepatic function were accompanied by reductions in circulating metabolites linked to hepatic insulin resistance (e.g., diacylglycerols, ceramides) in the Pharm TCR group.ConclusionsThe Pharm-TCR intervention did not improve fasting indices of beta-cell stress; however, markers of liver fat accumulation and and liver function were improved, suggesting that a LCER diet can improve some aspects of the underlying pathophysiology of T2D.Trial registrationClinicaltrials.gov (NCT03181165).

Chronic exposure to incretin metabolites GLP-1(9−36) and GIP(3−42) affect islet morphology and beta cell health in high fat fed mice

The incretin hormones, glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP), are rapidly degraded by dipeptidyl peptidase-4 (DPP-4) to their major circulating metabolites GLP-1(9−36) and GIP(3−42). This study investigates the possible effects of these metabolites, and the equivalent exendin molecule Ex(9−39), on pancreatic islet morphology and constituent alpha and beta cells in high-fat diet (HFD) fed mice. Male Swiss TO-mice (6–8 weeks-old) were maintained on a HFD or normal diet (ND) for 4 months and then received twice-daily subcutaneous injections of GLP-1(9−36), GIP(3−42), Ex(9−39) (25 nmol/kg bw) or saline vehicle (0.9% (w/v) NaCl) over a 60-day period. Metabolic parameters were monitored and excised pancreatic tissues were used for immunohistochemical analysis. Body weight and assessed metabolic indices were not changed by peptide administration. GLP-1(9−36) significantly (p<0.001) increased islet density per mm2 tissue, that was decreased (p<0.05) by HFD. Islet, beta and alpha cell areas were increased (p<0.01) following HFD and subsequently reduced (p<0.01-p<0.001) by GIP(3−42) and Ex(9−39) treatment. While GLP-1(9−36) did not affect islet and beta cell areas in HFD mice, it significantly (p<0.01) decreased alpha cell area. Compared to ND and HFD mice, GIP(3−42) treatment significantly (p<0.05) increased beta cell proliferation. Whilst HFD increased (p<0.001) beta cell apoptosis, this was reduced (p<0.01-p<0.001) by both GLP-1(9−36) and GIP(3−42). These data indicate that the major circulating forms of GLP-1 and GIP, namely GLP-1(9−36) and GIP(3−42) previously considered largely inactive, may directly impact pancreatic morphology, with an important protective effect on beta cell health under conditions of beta cell stress.

Induction of islet autoimmunity to defective ribosomal product of the insulin gene as neoantigen after anti-cancer immunotherapy leading to autoimmune diabetes.

The autoimmune response in type 1 diabetes (T1D), in which the beta cells expressing aberrant or modified proteins are killed, resembles an effective antitumor response. Defective ribosomal protein products in tumors are targets of the anti-tumor immune response that is unleashed by immune checkpoint inhibitor (ICI) treatment in cancer patients. We recently described a defective ribosomal product of the insulin gene (INS-DRiP) that is expressed in stressed beta cells and targeted by diabetogenic T cells. T1D patient-derived INS-DRiP specific T cells can kill beta cells and are present in the insulitic lesion. T cells reactive to INS-DRiP epitopes are part of the normal T cell repertoire and are believed to be kept in check by immune regulation without causing autoimmunity. T cell autoreactivity was tested using a combinatorial HLA multimer technology measuring a range of epitopes of islet autoantigens and neoantigen INS-DRiP. INS-DRiP expression in human pancreas and insulinoma sections was tested by immunohistochemistry. Here we report the induction of islet autoimmunity to INS-DRiP and diabetes after ICI treatment and successful tumor remission. Following ICI treatment, T cells of the cancer patient were primed against INS-DRiP among other diabetogenic antigens, while there was no sign of autoimmunity to this neoantigen before ICI treatment. Next, we demonstrated the expression of INS-DRiP as neoantigen in both pancreatic islets and insulinoma by staining with a monoclonal antibody to INS-DRiP. These results bridge cancer and T1D as two sides of the same coin and point to neoantigen expression in normal islets and insulinoma that may serve as target of both islet autoimmunity and tumor-related autoimmunity.

Yunvjian decoction mitigates hyperglycemia in rats induced by a high-fat diet and streptozotocin via reducing oxidative stress in pancreatic beta cells

Ethnopharmacological relevanceYunvjian (YNJ), a traditional Chinese herbal formula first reported in Jing Yue Quan Shu, is commonly used in the clinical treatment of type 2 diabetes mellitus (T2DM). However, the mechanism by which YNJ affects T2DM remains unclear. Aim of the studyThis study aimed to assess the therapeutic effects of YNJ on T2DM and explore the potential mechanism involved. Materials and methodsHigh-performance liquid chromatography (HPLC) was used to identify the chemical compounds of YNJ. The anti-T2DM effects of YNJ were observed in a high-fat diet/streptozotocin induced rat model. The type 2 diabetic rats were prepared as follows: rats were fed a high-fat diet for four weeks and then intraperitoneally injected with a low dose (30 mg/kg) of streptozotocin. YNJ and the positive control metformin were used in these experiments. Biochemical assays were implemented to determine the fasting blood glucose, glucose tolerance, insulin sensitivity, serum lipid levels, and oxidative stress index of the pancreas. Hematoxylin-eosin (H&E) staining was used to assess histopathological alterations in the pancreas. The mechanism by which YNJ affects T2DM was evaluated in INS-1 cells treated with glucose and high sodium palmitate. YNJ-supplemented serum was used in these experiments. Methyl thiazolyl tetrazolium assays, enzyme-linked immunosorbent assays, Nile red staining, flow cytometric analysis, and Western blotting were used to assess apoptosis, insulin secretion, lipid accumulation, reactive oxygen species production, and protein levels. ResultsFive major compounds were identified in YNJ. In high-fat diet/streptozotocin-induced diabetic rats, YNJ-M notably decreased fasting blood glucose and lipid levels; ameliorated glucose tolerance, insulin sensitivity, and islet morphology; reduced Malondialdehyde levels; and restored superoxide dismutase activity in the pancreatic islets. Furthermore, the effect of YNJ-M was significantly greater than that of YNJ-L, and YNJ-H had little effect on diabetic rats. In vitro experiments revealed that YNJ-supplemented serum (10%, 15%, and 20%) dramatically suppressed apoptosis, mitigated intracellular lipid accumulation and reduced intracellular oxidative stress levels in a dose-dependent manner. Additionally, YNJ-supplemented serum increased the protein expression of Nuclear factor erythroid 2-related factor 2, Heme oxygenase-1, and superoxide dismutase 1 and inhibited the protein expression of Kelch-like ECH-associated protein 1. ConclusionYNJ ameliorates high-fat diet/streptozotocin induced experimental T2DM. The underlying mechanism involves reducing oxidative stress in pancreatic beta cells. The findings of this study provide scientific justification for the application of the traditional medicine YNJ in treating T2DM.

Abstract B049: Pancreatic beta cell stress pathways drive pancreatic ductal adenocarcinoma development in obesity

Abstract Epidemiologic studies have shown that obesity is a risk factor for more than a dozen cancers, including pancreatic ductal adenocarcinoma (PDAC). As PDAC is one of the leading causes of cancer-related death and obesity rates are rapidly increasing worldwide, it is imperative that we understand the mechanisms by which obesity promotes PDAC progression. To address this gap in knowledge, our lab combined a well-established genetic model for obesity (O: Lepob/ob) with a Kras-driven pancreatic cancer model (KC: Pdx1-Cre; KrasLSL-G12D/+) that mimics human PDAC progression. Resultant KCO mice had a drastic increase in tumor development compared to their lean counterparts, whereas induced weight loss intercepted tumor progression, showing that the tumor-promoting effects of obesity can be reversed. Bulk RNA sequencing of tumors from these mice revealed aberrantly increased expression of multiple peptide hormones expressed in insulin-producing beta (b) cells of pancreatic islets, including cholecystokinin (CCK) and amylin (Iapp). Strikingly, transgenic expression of CCK and Iapp in b-cells was sufficient to promote Kras-driven pancreatic tumorigenesis in non-obese mice. Single-cell RNA sequencing of b cells showed a trajectory of changes in gene expression with increasing obesity from wildtype to high-fat diet-fed to Lepob/ob mice. Further in silico analysis combining archetypal (AANet) with pseudotime (TrajectoryNet) methods demonstrated that b cells misexpressing hormones (CCK and Iapp) arose from a specific subtype of preexisting b cells, specifically virgin b cells, a rare, immature, regenerative population. Preliminary in vivo lineage tracing analyses in MIP-CreERT;LSL-TdTomato;Lepob/ob mice support these findings. Finally, we investigated the transcriptional networks that lead to aberrant CCK and Iapp expression in b cells. By combining Grainger causality analysis with known gene-gene relationships in the Transcriptional Regulatory Relationship Unraveled by Sentence-based Text mining (TRRUST) v2 database, we discovered a stress-induced mitogen-activated protein kinase (MAPK8)-cJun pathway that promotes pro-tumorigenic hormone expression in b cells. These combined experimental and computational methods nominate novel b cell targets to subvert the development of PDAC, an exocrine tumor. Citation Format: Cathy C. Garcia, Aarthi Venkat, Alex Tong, Sherry Agabiti, Lauren Lawres, Rebecca Cardone, Richard Kibbey, Smita Krishnaswamy, Mandar D Muzumdar. Pancreatic beta cell stress pathways drive pancreatic ductal adenocarcinoma development in obesity [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B049.

SORCS2 activity in pancreatic α-cells safeguards insulin granule formation and release from glucose-stressed β-cells

Sorting receptor SORCS2 is a stress-response factor protecting neurons from acute insults, such as during epilepsy. SORCS2 is also expressed in the pancreas, yet its action in this tissue remains unknown. Combining metabolic studies in SORCS2-deficient mice with exvivo functional analyses and single-cell transcriptomics of pancreatic tissues, we identified a role for SORCS2 in protective stress response in pancreatic islets, essential to sustain insulin release. We show that SORCS2 is predominantly expressed in isletalpha cells. Loss of expression coincides with inability of these cells to produce osteopontin, a secreted factor that facilitates insulin release from stressed beta cells. In line with diminished osteopontin levels, beta cells in SORCS2-deficient islets show gene expression patterns indicative of aggravated cell stress, and exhibit defects in insulin granule maturation and a blunted glucose response. These findings corroborate a function for SORCS2 in protective stress response that extends to metabolism.

The amphiregulin/EGFR axis has limited contribution in controlling autoimmune diabetes

Conventional immunosuppressive functions of CD4+Foxp3+ regulatory T cells (Tregs) in type 1 diabetes (T1D) pathogenesis have been well described, but whether Tregs have additional non-immunological functions supporting tissue homeostasis in pancreatic islets is unknown. Within the last decade novel tissue repair functions have been ascribed to Tregs. One function is production of the epidermal growth factor receptor (EGFR) ligand, amphiregulin, which promotes tissue repair in response to inflammatory or mechanical tissue injury. However, whether such pathways are engaged during autoimmune diabetes and promote tissue repair is undetermined. Previously, we observed that upregulation of amphiregulin at the transcriptional level was associated with functional Treg populations in the non-obese diabetic (NOD) mouse model of T1D. From this we postulated that amphiregulin promoted islet tissue repair and slowed the progression of diabetes in NOD mice. Here, we report that islet-infiltrating Tregs have increased capacity to produce amphiregulin, and that both Tregs and beta cells express EGFR. Moreover, we show that amphiregulin can directly modulate mediators of endoplasmic reticulum stress in beta cells. Despite this, NOD amphiregulin deficient mice showed no acceleration of spontaneous autoimmune diabetes. Taken together, the data suggest that the ability for amphiregulin to affect the progression of autoimmune diabetes is limited.

Lupenone attenuates thapsigargin-induced endoplasmic reticulum stress and apoptosis in pancreatic beta cells possibly through inhibition of protein tyrosine kinase 2 activity

AimsProlonged high levels of cytokines, glucose, or free fatty acids are associated with diabetes, elevation of cytosolic Ca2+ concentration ([Ca2+]C), and depletion of Ca2+ concentration in the endoplasmic reticulum (ER) of pancreatic beta cells. This Ca2+ imbalance induces ER stress and apoptosis. Lupenone, a lupan-type triterpenoid, is beneficial in diabetes; however, its mechanism of action is yet to be clarified. This study evaluated the protective mechanism of lupenone against thapsigargin-induced ER stress and apoptosis in pancreatic beta cells. Materials and methodsMIN6, INS-1, and native mouse islet cells were used. Western blot for protein expressions, measurement of [Ca2+]C, and in vivo glucose tolerance test were mainly performed. Key findingsThapsigargin increased the protein levels of cleaved caspase 3, cleaved PARP, and the phosphorylated form of JNK, ATF4, and CHOP. Thapsigargin increased the interaction between stromal interaction molecule1 (Stim1) and Orai1, enhancing store-operated calcium entry (SOCE). SOCE is further activated by protein tyrosine kinase 2 (Pyk2), which is Ca2+-dependent and phosphorylates the tyrosine residue at Y361 in Stim1. Lupenone inhibited thapsigargin-mediated Pyk2 activation, suppressed [Ca2+]C, ER stress, and apoptosis. Lupenone restored impaired glucose-stimulated insulin secretion effectuated by thapsigargin and glucose intolerance in a low-dose streptozotocin-induced diabetic mouse model. SignificanceThese results suggested that lupenone attenuated thapsigargin-induced ER stress and apoptosis by inhibiting SOCE; this may be due to the hindrance of Pyk2-mediated Stim1 tyrosine phosphorylation. In beta cells that are inevitably exposed to frequent [Ca2+]C elevation, the attenuation of abnormally high SOCE would be beneficial for their survival.

High proinsulin:C-peptide ratio identifies individuals with stage 2 type 1 diabetes at high risk for progression to clinical diagnosis and responses to teplizumab treatment.

Tractable precision biomarkers to identify immunotherapy responders are lacking in type 1 diabetes. We hypothesised that proinsulin:C-peptide (PI:C) ratios, a readout of beta cell stress, could provide insight into type 1 diabetes progression and responses to immunotherapy. In this post hoc analysis, proinsulin and C-peptide levels were determined in baseline serum samples from 63 participants with stage 2 type 1 diabetes in the longitudinal TrialNet Teplizumab Prevention Study (n=41 in the teplizumab arm; n=22 in the placebo arm). In addition, previously tested demographic, C-peptide, glucose and proinsulin data were used for the new data analyses. The ratio of intact (unprocessed) proinsulin to C-peptide was analysed and relationships with progression to stage 3 diabetes were investigated. Elevated baseline PI:C was strongly associated with more rapid progression of diabetes in both the placebo and teplizumab treatment groups, but teplizumab abrogated the impact of high pre-treatment PI:C on type 1 diabetes progression. Differential responses of drug treatment in those with high vs low PI:C ratios were independent of treatment effects of teplizumab on the PI:C ratio or on relevant immune cells. High pre-treatment PI:C identified individuals with stage 2 type 1 diabetes who were exhibiting rapid progression to stage 3 disease and who displayed benefit from teplizumab treatment. These data suggest that readouts of active disease, such as PI:C ratio, could serve to identify optimal candidates or timing for type 1 diabetes disease-modifying therapies.

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

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. 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). 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. 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.

Identification of Compounds Protecting Pancreatic Islets against Oxidative Stress using a 3D Pseudoislet Screening Platform.

Oxidative stress leads to a lower success rate of clinical islet transplantation. Here, FDA-approved compounds are screened for their potential to decrease oxidative stress and to protect or enhance pancreatic islet viability and function. Studies are performed on in vitro "pseudoislet" spheroids, which are pre-incubated with 1280 different compounds and subjected to oxidative stress. Cell viability and oxidative stress levels are determined using a high-throughput fluorescence microscopy pipeline. Initial screening on cell viability results in 59 candidates. The top ten candidates are subsequently screened for their potential to decrease induced oxidative stress, and eight compounds efficient reduction of induced oxidative stress in both alpha and beta cells by 25-50%. After further characterization, the compound sulfisoxazole is found to be the most capable of reducing oxidative stress, also at short pre-incubation times, which is validated in primary human islets, where low oxidative stress levels and islet function are maintained. This study shows an effective screening strategy with 3D cell aggregates based on cell viability and oxidative stress, which leads to the discovery of several compounds with antioxidant capacity. The top candidate, sulfisoxazole is effective after a 30 min pre-incubation, maintains baseline islet function, and may help alleviate oxidative stress in pancreatic islets.

A DESCRIPTIVE COMPARISON OF RESPONSE OF ORAL HYPOGLYCEMIC AGENTS AMONG T2DM IN A BACKDROP OF INSULIN RESISTANCE

Introduction:Different homeostatic models for the assessment of beta cell function in patients with insulin resistance in type 2 diabetes mellitus suggest that Dipeptidyl Peptidase (DPP-4) inhibitors cause less beta cell stress. Aims: The present study aimed to compare and contrast insulin resistance in two groups of patients taking oral hypoglycemic agents, DPP-4 plus metformin and glimepiride plus metformin, on the basis of fasting and postprandial c-peptide and insulin resistance estimated by homeostatic model assessment of insulin resistance (HOMA-IR). Methods: This preliminary descriptive observational study was conducted from 2018 to 2019 in the service Laboratory of the Department of Biochemistry, in collaboration with the Endocrinology Department, Nil Ratan Sircar Medical College and Hospital, Kolkata. Serum C-peptide, serum insulin, and plasma glucose levels were measured in both fasting and post-prandial states along with glycated hemoglobin. Result: In the fasting and fed state, the secretagogue effect of glimepiride-metformin combination was significantly higher (p = 0.017) than that of the linagliptin-metformin combination. Conclusion: Patients treated with glimepiride showed high post prandial insulin levels and high post prandial glucose excursion. This finding can be explained by the probable increase in insulin resistance, which is reflected in their post-prandial C peptide level. However, in the case of linagliptin, one mechanism of decreased post-prandial glucose is believed to be the inhibition of α-cell glucagon release, thereby relieving β-cell stress

Application of fluorescence lifetime imaging microscopy to monitor glucose metabolism in pancreatic islets in vivo.

Glucose stimulated insulin secretion is mediated by glucose metabolism via oxidative phosphorylation generating ATP that triggers membrane depolarization and exocytosis of insulin. In stressed beta cells, glucose metabolism is remodeled, with enhanced glycolysis uncoupled from oxidative phosphorylation, resulting in the impaired glucose-mediated insulin secretion characteristic of diabetes. Relative changes in glycolysis and oxidative phosphorylation can be monitored in living cells using the 3-component fitting approach of fluorescence lifetime imaging microscopy (FLIM). We engrafted pancreatic islets onto the iris to permit in vivo FLIM monitoring of the trajectory of glucose metabolism. The results show increased oxidative phosphorylation of islet cells (∼90% beta cells) in response to hyperglycemia; in contrast red blood cells traversing the islets maintained exclusive glycolysis as expected in the absence of mitochondria.

Donor noradrenaline use is associated with better allograft survival in recipients of pancreas transplantation.

Outcomes following pancreas transplantation are suboptimal and better donor selection is required to improve this. Vasoactive drugs (VaD) are commonly used to correct the abnormal haemodynamics of organ donors in intensive care units. VaDs can differentially affect insulin secretion positively (dobutamine) or negatively (noradrenaline). The hypothesis was that some VaDs might induce beta-cell stress or rest and therefore impact pancreas transplant outcomes. The aim of the study was to assess relationships between VaD use and pancreas transplant graft survival. Data from the UK Transplant Registry on all pancreas transplants performed between 2004 and 2016 with complete follow-up data were included. Univariable- and multivariable-adjusted Cox regression analyses determined risks of graft failure associated with VaD use. In 2,183 pancreas transplants, VaDs were used in the following numbers of donors: dobutamine 76 (3.5%), dopamine 84 (3.8%), adrenaline 161 (7.4%), noradrenaline 1,589 (72.8%) and vasopressin 1,219 (55.8%). In multivariable models, adjusted for covariates and the co-administration of other VaDs, noradrenaline use (vs non-use) was a strong predictor of better graft survival (hazard ratio [95% confidence interval] 0.77 [0.64-0.94], p = 0.01). Noradrenaline use was associated with better graft survival in models adjusted for donor and recipient variables - this may be related to inhibition of pancreatic insulin secretion initiating pancreatic beta-cell 'rest'. Further research is required to replicate these findings and establish whether relationships are causal. Identification of alternative methods of inducing beta-cell rest could be valuable in improving graft outcomes.

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

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

Benzophenones alter autophagy and ER stress gene expression in pancreatic beta cells in vitro

Benzophenones (BPs) are endocrine disruptors frequently used in sunscreens and food packaging as UV blockers. Our goal was to assess the effect of benzophenone 2 (BP2) and 3 (BP3) on gene expression related to autophagy process and ER stress response in pancreatic beta cells. To that end, the mouse pancreatic beta cell line MIN6B1 was treated with 10µM BP2 or BP3 in the presence or absence of the autophagy-inhibitor chloroquine (CQ, 10µM) or the autophagy-inducer rapamycin (RAPA, 50nM) during 24h. BP3 inhibited the expression of the autophagic gene Ulk1, and additional effects were uncovered when autophagy was modified by CQ and RAPA. BP3 counteracted CQ-induced Lamp2 expression but did not compensate CQ-induced Sqstm1/p62 gene transcription, neither BP2. Nevertheless, the BPs did not alter the autophagic flux. In relation to ER stress, BP3 inhibited unspliced and spliced Xbp1 mRNA levels in the presence or absence of CQ, totally counteracted CQ-induced Chop gene expression, and partially reverted CQ-induced Grp78/Bip mRNA levels, while BP2 also partially inhibited Grp78/Bip mRNA induction by CQ. In conclusion, BPs, principally BP3, affect cellular adaptive responses related to autophagy, lysosomal biogenesis, and ER stress in pancreatic beta cells, indicating that BP exposure could lead to beta cell dysfunction.