Insulin Secretion In Response Research Articles

MicroRNA 29 modulates β-cell mitochondrial metabolism and insulin secretion via underlying miR-29-OXPHOS complex pathways.

MicroRNAs (miRNAs) regulate β-cell function, and β-cell mitochondria and insulin secretion are perturbed in diabetes. We aimed to identify key miRNAs regulating β-cell mitochondrial metabolism and novel β-cell miRNA-mitochondrial pathways. TargetScan (http://www.targetscan.org/) was used to predict if 16 miRNAs implicated in β-cell function target 27 cis-eGenes implicated in mitochondrial activity. The expression of candidate miRNAs and insulin secretion after 24 and 1 h pre-incubation in 2.8, 11.1- and 16.7-mM glucose was measured in clonal INS-1 832/13 β-cells. MiR-29 silenced INS-1 832/13 cells were assessed for insulin secretion (glucose, pyruvate, and K+), target cis-eGene expression (Ndufv3 and Ndufa10 components of mitochondrial complex I (CI)), OXPHOS (CI-V) protein expression, and mitochondrial OXPHOS respiration/activity. The expression of differentially expressed miR-29 miRNAs was evaluated in Goto-Kakizaki (GK) rat, db/db mouse and type 2 diabetic (T2D) human islets, as well as NMRI mouse islets cultured under glucolipotoxic conditions. MiR-29, miR-15 and miR-124 were predicted to regulate ~20 cis-eGenes, while miR-29 alone was predicted to regulate ≥12 of these in rat and human species. MiR-29 expression and insulin secretion were reduced in INS-1 832/13 cells after 24 h in elevated glucose. MiR-29 knockdown increased all tested insulin secretory responses, Nudfv3, Ndufa10, complex I and II expression, and cellular mitochondrial OXPHOS. MiR-29 expression was reduced in db/db islets but increased in GK rat and T2D human islets. We conclude miR-29 is a key miRNA in regulating β-cell mitochondrial metabolism and insulin secretion via underlying miR-29-OXPHOS complex pathways. Furthermore, we infer reduced miR-29 expression compensatorily enhances insulin secretion under glucotoxicity.

Baseline phenotypes with preserved β-cell function and high insulin concentrations have the best improvements in glucose tolerance after weight loss: results from the prospective DEXLIFE and EGIR-RISC studies

BackgroundWeight loss and lifestyle intervention improve glucose tolerance delaying the onset of type 2 diabetes (T2D), but individual responses are highly variable. Determining the predictive factors linked to the beneficial effects of weight loss on glucose tolerance could provide tools for individualized prevention plans. Thus, the aim was to investigate the relationship between pre-intervention values of insulin sensitivity and secretion and the improvement in glucose metabolism after weight loss. MethodsIn the DEXLIFE cohort (373 individuals at high risk of T2D, assigned 3:1 to a 12-week lifestyle intervention or a control arm, Trial Registration: ISRCTN66987085), K-means clustering and logistic regression analysis were performed based on pre-intervention indices of insulin sensitivity, insulin secretion (AUC-I), and glucose-stimulated insulin response (ratio of incremental areas of insulin and glucose, iAUC I/G). The response to the intervention was evaluated in terms of reduction of OGTT-glucose concentration. Clusters' validation was done in the prospective EGIR-RISC cohort (n = 1538). ResultsFour replicable clusters with different glycemic and metabolomic profiles were identified. Individuals had similar weight loss, but improvement in glycemic profile and β-cell function was different among clusters, highly depending on pre-intervention insulin response to OGTT. Pre-intervention high insulin response was associated with the best improvement in AUC-G, while clusters with low AUC-I and iAUC I/G showed no beneficial effect of weight loss on glucose control, as also confirmed by the logistic regression model. ConclusionsIndividuals with preserved β-cell function and high insulin concentrations at baseline have the best improvement in glucose tolerance after weight loss.

Interplay of Transcriptional Factors In Beta Cells Development and Maturation

Diabetes is a group of metabolic disorders resulting from defects in insulin secretion, insulin action, or both. It is characterized by high blood sugar levels over a prolonged period of time with disturbances of carbohydrate, protein, and fat metabolism. It is a global disorder affecting about half a billion or 536.6 million people worldwide, which is said to rise to 783.2 million by 2045. To maintain normoglycemia, pancreatic β-cells release insulin in proportion to the amounts of nutrients in the blood. Through a process of postnatal development, β-cells learn to connect insulin release to food cues. The insulin secretory response in mature β-cells adjusts to alterations in nutritional status. The interaction between transcriptional programs specific to each cell type and external stimuli is necessary for both β-cell maturation and functional adaptation. In this review, we examine the growing data that suggests lineage-determining and signal-dependent transcription factors (LDTFs and SDTFs, respectively) work together to regulate β-cell activity during development and homeostasis. In-depth knowledge of β-cell SDTFs and their corresponding signals would clarify the processes involved in β-cell maturation and functional adaptation, directly affecting diabetes treatments and the production of mature β-cells from stem cells.

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

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

Insulin Secretion in Extended Mixed-Meal Tolerance Test in the Context of the Four Phases of Digestion

Background: A sequential response of insulin secretion is likely to follow the sequential pattern of digestion to optimize the effect of insulin and other counter regulatory peptides on the target tissues to maintain optimum insulin signaling pathway activation. Therefore, it is likely to relate insulin secretory response to the corresponding phase of digestion. Patients and methods: Plasma glucose, insulin and glucagon levels of three groups, each comprises of 10 persons. Group “N” for normal, group “P” for pre-diabetic and group “D” for type 2 diabetes individuals. Fluctuations in insulin secretion, in relation to changes of plasma glucose levels throughout the four phases of digestion will be traced, changes in glucagon levels will be also evaluated. Results: Early in the gastric-filling phase insulin was decreasing while glucose was rising, whereas, early in the gastric-emptying phase, insulin was rising while glucose was decreasing. Statistical significance of glucagon changes was higher than that of insulin between groups. Conclusion: Factors, which regulate insulin secretion during the gastric-filling phase, may differ than those during the gastric emptying-phase. Insulin/Glucagon ratio, is more contributed, than insulin alone, to the metabolic abnormalities associated with diabetes. It is more informative to consider insulin secretion in relation to the corresponding phase of digestion. Therefore, a four- phase cycle of insulin secretion may be suggested.

Insulin release from isolated cat islets of Langerhans

Feline diabetes mellitus is a common endocrine disease with increasing prevalence. It shows similarities with human type 2 diabetes and is characterized by insulin resistance and deficient insulin secretion. Moreover, cats and humans belong to the very few species that form amyloid depositions in the pancreatic islets. However, little is known about cat islet function and no studies have addressed insulin secretion from isolated islets ex vivo. The aim of this study was to establish a protocol for isolation of islets of Langerhans from pancreata of cats euthanized due to disease, and to evaluate insulin secretion responses to various physiological and pharmacological stimuli. Collagenase digestion of pancreatic tissue from 13 non-diabetic cats and two cats with diabetic ketoacidosis yielded individual islets surrounded by a layer of exocrine tissue that was reduced after two days in culture. Histological examination showed islet amyloid in pancreatic biopsies from most non-diabetic and in one diabetic cat. Islets from non-diabetic cats cultured at 5.5 mM glucose responded with increased insulin secretion to 16.7 mM glucose, 30 mM K+ and 20 µM of the sulfonylurea glipizide (2-3 times basal secretion at 3 mM glucose). The glucagon-like peptide-1 receptor agonist exendin-4 (100 nM) had no effect under basal conditions but potentiated glucose-triggered insulin release. Only one of nine islet batches from diabetic cats released detectable amounts of insulin, which was enhanced by exendin-4. Culture of islets from non-diabetic cats at 25 mM glucose impaired secretion both in response to glucose and K+ depolarization. In conclusion, we describe a procedure for isolation of islets from cat pancreas biopsies and demonstrate that isolated cat islets secrete insulin in response to glucose and antidiabetic drugs. The study provides a basis for future ex vivo studies of islet function relevant to the understanding of the pathophysiology and treatment of feline diabetes.

Absence of Slc39a14/Zip14 in mouse pancreatic beta cells results in hyperinsulinemia.

Zinc is an essential component of the insulin protein complex synthesized in β cells. The intracellular compartmentalization and distribution of zinc are controlled by 24 transmembrane zinc transporters belonging to the ZnT or Zrt/Irt-like protein (ZIP) family. Downregulation of SLC39A14/ZIP14 has been reported in pancreatic islets of patients with type 2 diabetes (T2D) as well as mouse models of high-fat diet (HFD)- or db/db-induced obesity. Our previous studies observed mild hyperinsulinemia in mice with whole body knockout of Slc39a14 (Zip14 KO). Based on our current secondary data analysis from an integrative single-cell RNA-seq dataset of human whole pancreatic tissue, SLC39A14 (coding ZIP14) is the only other zinc transporter expressed abundantly in human β cells besides well-known zinc transporter SLC30A8 (coding ZnT8). In the present work, using pancreatic β cell-specific knockout of Slc39a14 (β-Zip14 KO), we investigated the role of SLC39A14/ZIP14-mediated intracellular zinc trafficking in glucose-stimulated insulin secretion and subsequent metabolic responses. Glucose-stimulated insulin secretion, zinc concentrations, and cellular localization of ZIP14 were assessed using in vivo, ex vivo, and in vitro assays using β-Zip14 KO, isolated islets, and murine cell line MIN6. Metabolic evaluations were done on both chow- and HFD-fed mice using time-domain nuclear magnetic resonance and a comprehensive laboratory animal monitoring system. ZIP14 localizes on the endoplasmic reticulum regulating intracellular zinc trafficking in β cells and serves as a negative regulator of glucose-stimulated insulin secretion. Deletion of Zip14 resulted in greater glucose-stimulated insulin secretion, increased energy expenditure, and shifted energy metabolism toward fatty acid utilization. HFD caused β-Zip14 KO mice to develop greater islet hyperplasia, compensatory hyperinsulinemia, and mild insulin resistance and hyperglycemia. This study provided new insights into the contribution of metal transporter ZIP14-mediated intracellular zinc trafficking in glucose-stimulated insulin secretion and subsequent metabolic responses.NEW & NOTEWORTHY Metal transporter SLC39A14/ZIP14 is downregulated in pancreatic islets of patients with T2D and mouse models of HFD- or db/db-induced obesity. However, the function of ZIP14-mediated intracellular zinc trafficking in β cells is unknown. Our analyses revealed that SLC39A14 is the only Zn transporter expressed abundantly in human β cells besides SLC30A8. Within the β cells, ZIP14 is localized on the endoplasmic reticulum and serves as a negative regulator of insulin secretion, providing a potential therapeutic target for T2D.

SAT064 Establishment And Initial Characterization Of Two Parallel Targeted Human ZnT8 Knock-in Murine Lines For Studying The Type 2 Diabetes Risk Variant At SNP rs13266634 Of The ZnT8 Gene

Abstract Disclosure: J.C. Wang: None. C. Adelma: None. M.G. Hayes: None. M. El Muayed: None. Background: Studies showed an association between R variant at position 325 in the β-cell zinc transporter ZnT8 and an increased risk for developing type 2 diabetes mellitus (T2DM). However, the mechanism by which this variant confers an increased risk for type 2 diabetes remains unclear. Methods: We generated a murine model for studying the effect of SNP rs13266634 on β-cell physiology. This model consists of two parallel murine lines in which conditional (RIP-Cre induced) expression constructs for the human variants of SLC30A8 (ZnT8), namely R (hZnT8R325, T2DM risk allele) and W (hZnT8W325, non-risk allele) of SNPrs13266634 were inserted into the rosa26 locus via targeted integration. β-cell specific expression of each of ZnT8R325 and ZnT8W325 was induced by cross breeding with a RIP-Cre line, expressing Cre recombinase in β-cells. All mouse lines were backcrossed into a C57BL/6N background null for the mouse variant of ZnT8. Islet zinc concentrations was measured by ICP-MS following hydrolysis in HNO3. Ex-vivo Glucose Stimulated Insulin Secretion Ratio (GSISR) was measured by incubation of isolated islets in 2.8 mM glucose, followed by 16.7 mM glucose minutes after an initial 60 min equilibration phase and calculating the insulin secretion at 16.7 mM/2.8 mM glucose. Data are reported as averages ± SEM from 3 independent experiments (n=3), each with 4 technical replicates. Results: Expression of both variants was verified by mRNA PCR from islets. Levels of expression in both lines were identical, owing to the targeted integration approach. Expression of each of hZnT8R325 and ZnT8W325 resulted in restoration of islet zinc concentration to levels similar to those found in normal human islets and wild type mouse islets. This confirms proper function of the hZnT8R325 transgenes in this newly established model. hZnT8R expressing islets showed a trend to lower insulin response to ex-vivo glucose stimulation compared to hZnT8W325 expressing islets (GSISR- 1.37 ± 0.41 vs 1.97 ± 0.32 respectively), as well as Cre expressing (GSISR 1.53 ± 0.40) or inactive construct controls (GSISR 2.43 ± 0.57). In-vivo glucose stimulated insulin secretion was not significantly different between hZnT8R325 expressing mice and those expressing hZnT8W325. As previously reported by others, all mice with β-cell Cre expression (RIP-Cre activated hZnT8 expressing lines and RIP-Cre controls) showed a lower insulin secretory response to glucose compared to non-Cre expressing mice (inactive construct controls). Conclusion: The newly created murine mouse model expresses functional human homologues of the ZnT8 variants hZnT8R325 and hZnT8W325 that result in restored beta cell zinc uptake. A trend to lower insulin secretory capacity was observed in ex-vivo glucose stimulated insulin secretion experiments. This model will be valuable tool to elucidate the functional consequences of the T2DM risk variant at SNP rs13266634. Presentation: Saturday, June 17, 2023

SAT062 Inhibition Of GIPR Expression Enhances Sulfonylurea-induced Insulin Secretion, And It Was Associated With Changes In ATP Contents

Abstract Disclosure: H. Kim: None. J. Lim: None. H. Jung: None. Introduction: Sulfonylurea (SU) might be indispensable for a subset of patients with type 2 diabetes. In our previous study, we collected some patients who were extremely sensitive to SU for optimal glycemic control, then drew several candidate single nucleotide variants (SNV) using whole exome sequencing. Among them, 3 SNV (rs550405192, rs13306403, and rs13306402) were harbored by GIPR, coding a receptor for gastric inhibitory polypeptide 1-2). Therefore, in vitro study of GIPR on insulin secretory response to SU was performed in this study. Method:Gipr silencing was done in INS-1 cells using siRNA transfection. To induce glucolipotoxicity, 15-mM glucose and 150-uM palmitic acid were treated to the cells for 24 hours. Glimepiride (50 nM) was treated for 30 min in KRBH buffer with low-/high-glucose, following low-glucose starvation. Intracellular ATP contents and released ATP were measured with a bioluminescence assay kit. Quantitative RT-PCR to assess Vdac1 expression. Results:Gipr silencing was confirmed by reduced expression of mRNA and protein. GIP-induced insulin secretion was also down-regulated. Gipr silencing did not affect either basal or glucose-stimulated insulin secretion. As for SU-induced insulin secretion, Gipr silencing significantly increased it in a low-glucose condition (2.8 mM) but not in a high-glucose (17.5 mM). However, after chronic exposure to glucolipotoxicity, Gipr silencing significantly enhanced the response to SU in a high-glucose condition, too. These effects of Gipr silencing on the SU response were associated with changes in cellular ATP contents, which is critical to SU effects. Because diabetic condition had been reported to induce Vdac1 expression and subsequent ATP release in insulin-secreting cells3), we examined them with regard to Gipr silencing. Vdac1 expression was significantly increased when exposed to glucolipotoxicity, and Gipr silencing inhibited both Vdac1 expression and ATP release in the condition. Conclusion: According to the suppression of Vdac1 expression and ATP release by Gipr silencing in INS1 cells under glucolipotoxicity, interference of GIPR signaling would enhance SU-induced insulin secretion by preservation of cellular ATP. This study supported that loss-of-function mutations in GIPR as novel candidates regarding to sensitivity to SU in type 2 diabetes.

SAT074 Induction Of Insulin Hypersecretion Uncovers Distinctions Between Adaptive And Maladaptive Endoplasmic Reticulum Stress Response In Beta Cells

Abstract Disclosure: G. Roy: None. K. Rodrigues dos Santos: None. M.B. Kwakye: None. Z. Tan: None. T.S. Johnson: None. M.A. Kalwat: None. Pancreatic islet β-cells release insulin to maintain glucose homeostasis. β-cells must translate, package, and secrete large amounts of insulin. During this process the unfolded protein response of the endoplasmic reticulum (UPRER) is induced to maintain these functions. However, stimuli that force β-cell to secrete insulin at enhanced rates and for prolonged durations risk inducing the terminal UPRER and eventual apoptosis. In a chemical screen for insulin secretion modulators, we discovered SW016789 which caused hypersecretion of insulin and led to a transient induction of the UPRER, but not apoptosis. In contrast, SERCA2 ER Ca2+ pump inhibitor thapsigargin induces the terminal UPRER. We hypothesized that SW016789 can be used as a tool compound to discover genes involved in β-cell adaptation to hypersecretion-induced stress. We performed time course transcriptomics in MIN6 β-cells exposed to SW016789 (5 µM) or thapsigargin (100 nM) from 0-24 h. Unbiased analyses using a Dirichlet process Gaussian process (DPGP) method revealed clusters of genes temporally co-regulated and the genes within these clusters were distinct between SW016789 and thapsigargin treatments. In particular, after 6 h of SW016789-induced hypersecretion we found a highly induced cluster of genes (SW cluster 3) enriched in adaptive UPRER factors (e.g. Manf). Conversely, most of the thapsigargin-induced genes clustered at 24 h and were enriched for terminal UPRER factors (e.g. Txnip). Pathway analysis of SW cluster 3 indicated that genes involved in in regulation of mRNA methylation and ER-associated degradation were also induced by SW016789 sooner and with greater amplitude than by thapsigargin, suggesting distinct differences in the handling of protein translation and degradation. From the SW cluster 3 genes we selected proteins known to be ER-associated or secreted and generated stable transgenic or CRISPR knockout MIN6 β-cell lines for each. Our data suggest altered expression of these factors may impair glucose-stimulated insulin secretion responses and alter cell viability in presence or absence of ER stressors including cytokines, thapsigargin, and tunicamycin. In conclusion, we have successfully shown that pharmacological induction of insulin hypersecretion can induce a distinct transcriptional outcome from that of canonically-induced UPRER and that we can take advantage of this property to discover new β-cell regulatory pathways and targets. We envision this dataset as a resource for the secretory biology and islet biology communities. Presentation: Saturday, June 17, 2023

Diabetes Mellitus: A Review and Update

Diabetes mellitus is a group of metabolic diseases which are typified by the development of hyperglycaemia which do emanate from defects in insulin secretion, insulin action, or both. The chronic hyperglycaemia of diabetes is associated with long-term damage, dysfunction, and failure of various organs, especially the eyes, kidneys, nerves, heart, and blood vessels. Various types of diabetes mellitus exist which have been discussed in the main part of this article. Many pathogenic processes are involved in the development of diabetes mellitus. These pathogenic processes range from autoimmune destruction of the beta-cells of the pancreas with consequent insulin deficiency to abnormalities which result in resistance to insulin action. The basis of the abnormalities in carbohydrate, fat, and protein metabolism in diabetes is deficient action of insulin on target tissues. Deficient insulin action ensues inadequate insulin secretion and / or diminished tissue responses to insulin at one or more points within the complex pathways of hormone action. Impairment of insulin secretion and defects in insulin action frequently coexist in the same patient, and it is often unclear which abnormality, if either alone, is the primary cause of the hyperglycaemia. The manifestations of marked hyperglycaemia do include: polyuria, polydipsia, weight loss, sometimes with polyphagia, and blurred vision. Impairment of growth and susceptibility to certain infections may also ensue chronic hyperglycaemia. Acute, life-threatening consequences of diabetes mellitus include: hyperglycaemia with ketoacidosis or the nonketotic hyperosmolar syndrome. Long-term complications of diabetes mellitus do include: diabetic retinopathy with potential loss of vision; diabetic nephropathy which leads to kidney (renal) failure; peripheral neuropathy with risk of foot ulcers, amputation, and Charcot joints; as well as autonomic neuropathy which cause gastrointestinal, genitourinary, as well as cardiovascular symptoms and sexual dysfunction. Glycation of tissue proteins and other macromolecules and excess production of polyol compounds from glucose are among the mechanisms that had been postulated to produce tissue damage from chronic hyperglycaemia. Patients who have diabetes mellitus do have an increased incidence of atherosclerotic cardiovascular, peripheral vascular, and cerebrovascular disease. Hypertension, abnormalities of lipoprotein metabolism, and periodontal disease are often found in people who have diabetes mellitus. The emotional and social impact of diabetes mellitus as well as the demands of treatment of diabetes and its complications could cause significant psychosocial dysfunction in patients who have diabetes mellitus as well as their families. Even though there are many types of diabetes mellitus, the vast majority of cases of diabetes mellitus do fall into two broad etiopathogenetic categories which have been extensively discussed in the ensuing article below that has been divided into (a) Overview and (b) miscellaneous narrations. In one category of diabetes mellitus type 1 diabetes mellitus, the cause is an absolute deficiency of insulin secretion. Individuals at increased risk of developing this type of diabetes could often be identified by serological evidence of an autoimmune pathological process that occur within the pancreatic islets and by genetic markers. In the other type of diabetes mellitus, which is the much more prevalent category that is referred to as type 2 diabetes mellitus, the cause of the disease is a combination of resistance to insulin action and an inadequate compensatory insulin secretory response. In the latter category, a degree of hyperglycaemia sufficient to cause pathological and functional changes in various target tissues, but without clinical symptoms, could be present for a long period of time before diabetes mellitus is diagnosed. During this asymptomatic period, it is possible to demonstrate an abnormality in carbohydrate metabolism by measurement of plasma glucose in the fasting state or after a challenge with an oral glucose load. Considering that diabetes mellitus is common as well as the symptoms of diabetes mellitus are non-specific symptoms that may simulate diabetes mellitus, a number of individuals who are afflicted by diabetes mellitus do not know they have diabetes mellitus, there is need for a global educational program on the manifestations and approach that is needed for early diagnosis of the disease so that all clinicians and the entire global population would have a high index of suspicion of the disease. There also a global life style education including regular exercise that would delay onset of or reduce the severity of type 2 diabetes and to improve the quality of life of patients who have diabetes mellitus. It is important to dedicate time to read the article carefully which contains documentations related to the World Health Organization’s global effort to reduce the incidence and severity of diabetes mellitus and all individuals globally should follow carefully recommendations of the World Health organization as well as recommendations of other organizations in the world that have devoted their time to health education on diabetes mellitus. and a number of people

A pathway model of glucose-stimulated insulin secretion in the pancreatic β-cell.

The pancreas plays a critical role in maintaining glucose homeostasis through the secretion of hormones from the islets of Langerhans. Glucose-stimulated insulin secretion (GSIS) by the pancreatic β-cell is the main mechanism for reducing elevated plasma glucose. Here we present a systematic modeling workflow for the development of kinetic pathway models using the Systems Biology Markup Language (SBML). Steps include retrieval of information from databases, curation of experimental and clinical data for model calibration and validation, integration of heterogeneous data including absolute and relative measurements, unit normalization, data normalization, and model annotation. An important factor was the reproducibility and exchangeability of the model, which allowed the use of various existing tools. The workflow was applied to construct a novel data-driven kinetic model of GSIS in the pancreatic β-cell based on experimental and clinical data from 39 studies spanning 50 years of pancreatic, islet, and β-cell research in humans, rats, mice, and cell lines. The model consists of detailed glycolysis and phenomenological equations for insulin secretion coupled to cellular energy state, ATP dynamics and (ATP/ADP ratio). Key findings of our work are that in GSIS there is a glucose-dependent increase in almost all intermediates of glycolysis. This increase in glycolytic metabolites is accompanied by an increase in energy metabolites, especially ATP and NADH. One of the few decreasing metabolites is ADP, which, in combination with the increase in ATP, results in a large increase in ATP/ADP ratios in the β-cell with increasing glucose. Insulin secretion is dependent on ATP/ADP, resulting in glucose-stimulated insulin secretion. The observed glucose-dependent increase in glycolytic intermediates and the resulting change in ATP/ADP ratios and insulin secretion is a robust phenomenon observed across data sets, experimental systems and species. Model predictions of the glucose-dependent response of glycolytic intermediates and biphasic insulin secretion are in good agreement with experimental measurements. Our model predicts that factors affecting ATP consumption, ATP formation, hexokinase, phosphofructokinase, and ATP/ADP-dependent insulin secretion have a major effect on GSIS. In conclusion, we have developed and applied a systematic modeling workflow for pathway models that allowed us to gain insight into key mechanisms in GSIS in the pancreatic β-cell.

286-OR: Using iPSC-Derived Islets to Model the Effect of an ABCC8 Arg1420His Mutation in Fetal and Adult Islets

We previously reported an Arg1420His mutation in ABCC8 in an Indigenous community that resulted in hyperinsulinemic hypoglycemia in infancy in a homozygous carrier and a doubling in type 2 diabetes (T2D) risk in heterozygous carriers (3% of the population). Previous studies of ABCC8 mutations have suggested a loss of pancreatic beta cells as a probable cause of future T2D. To understand the pathomechanism behind the increased risk of T2D due to the Arg1420His mutation, we generated two sets of isogenic iPSCs from two different Indigenous community members. These iPSCs were used to generate pancreatic islets and assess insulin secretion using a dynamic glucose stimulated insulin secretion (D-GSIS) assay when the islets were immature (resembling fetal islets) and during multiple days after maturation (resembling adult islets). In immature islets, the D-GSIS assay confirmed the clinical phenotype of significantly higher insulin secretion in islets with the His-allele under basal conditions (2mM glucose) and a lower response to diazoxide in islets homozygous for the His-allele. Both Arg1420 and His1420 mature islets had lower insulin secretion under basal conditions, however, insulin secretory response to high glucose (20mM) and Tolbutamide (sulphonylurea) was significantly lower in islets heterozygous for the His-allele whereas no response was seen in islets homozygous for the His-allele despite having similar proportion of beta cells as assessed by flow cytometry (~50% cells positive for INS and NKX6-1). This suggests a mechanism other than beta cell loss as the cause of lower insulin secretory response in these impaired islets. Single cell RNA sequencing showed higher expression of G6PC2, which inhibits insulin secretion by opposing the action of GCK, in islets with the His-allele. Our ongoing work includes knockout of G6PC2 in the isogenic iPSCs and using a GCK activator (Dorzagliatin) to correct the insulin secretory response to high glucose in the His-allele carrying islets. Disclosure A.K.Nair: None. K.Barkho: None. M.Traurig: None. R.G.Nelson: None. C.Bogardus: None. L.Baier: None. Funding National Institute of Diabetes and Digestive and Kidney Diseases

Improved Insulin Secretion Response and Beta-cell Function Correlated with Increased Prolactin Levels After Laparoscopic Sleeve Gastrectomy in Morbidly Obese Patients with Acanthosis Nigricans.

To explore insulin secretion patterns, β-cell function, and serum prolactin (PRL) concentrations in Chinese morbidly obese patients with Acanthosis nigricans (AN) and their alterations after laparoscopic sleeve gastrectomy (LSG). A total of 138 morbidly obese subjects undergoing LSG were categorized as simple obesity without AN (OB group, n = 55) and obesity with AN (AN group, n = 83). Oral glucose tolerance test (OGTT), PRL, and related metabolic indices were performed pre- and 12 months post-LSG. Insulin secretion patterns were derived from insulin secretion peak time during OGTT: type I (peak at 30 or 60 min) and type II (peak at 120 or 180 min). Preoperatively, AN group showed significantly higher proportions of type II insulin secretion pattern, fasting insulin (FINS), and homeostatic model assessment of insulin resistance (HOMA-IR) whereas lower oral glucose insulin sensitivity (OGIS), insulinogenic index (IGI), and disposition index (DI) than OB group, which were improved significantly at 12 months postoperatively in both groups, more pronounced in AN group. Intriguingly, serum PRL declined substantially in AN group than OB group at baseline whereas elevated only in the AN group post-LSG. After adjusting for confounding factors, elevated PRL correlated significantly with increased IGI and DI, and decreased HOMA-IR in both genders, as well as increased OGIS in females, which was detected only in the AN group CONCLUSION: Morbidly obese patients with AN presented delayed insulin secretion response, impaired insulin secretion, and β-cell dysfunction, which were significantly improved by LSG and might benefit from elevated PRL.

Decellularized bladder as scaffold to support proliferation and functionality of insulin-secreting pancreatic cells.

Loss in the number or function of insulin-producing β-cells in pancreatic islets has been associated with diabetes mellitus. Although islet transplantation can be an alternative treatment, complications such as apoptosis, ischaemia and loss of viability have been reported. The use of decellularized organs as scaffolds in tissue engineering is of interest owing to the unique ultrastructure and composition of the extracellular matrix (ECM) believed to act on tissue regeneration. In this study, a cell culture system has been designed to study the effect of decellularized porcine bladder pieces on INS-1 cells, a cell line secreting insulin in response to glucose stimulation. Porcine bladders were decellularized using two techniques: a detergent-containing and a detergent-free methods. The resulting ECMs were characterized for the removal of both cells and dsDNA. INS-1 cells were not viable on ECM produced using detergent (i.e., sodium dodecyl sulfate). INS-1 cells were visualized following 7 days of culture on detergent-free decellularized bladders using a cell viability and metabolism assay (MTT) and cell proliferation quantified (CyQUANT™ NF Cell Proliferation Assay). Further, glucose-stimulated insulin secretion and immunostaining confirmed that cells were functional in response to glucose stimulation, as well as they expressed insulin and interacted with the detergent-free produced ECM, respectively.

A multicentric consortium study demonstrates that dimethylarginine dimethylaminohydrolase 2 is not a dimethylarginine dimethylaminohydrolase

Dimethylarginine dimethylaminohydrolase 1 (DDAH1) protects against cardiovascular disease by metabolising the risk factor asymmetric dimethylarginine (ADMA). However, the question whether the second DDAH isoform, DDAH2, directly metabolises ADMA has remained unanswered. Consequently, it is still unclear if DDAH2 may be a potential target for ADMA-lowering therapies or if drug development efforts should focus on DDAH2’s known physiological functions in mitochondrial fission, angiogenesis, vascular remodelling, insulin secretion, and immune responses. Here, an international consortium of research groups set out to address this question using in silico, in vitro, cell culture, and murine models. The findings uniformly demonstrate that DDAH2 is incapable of metabolising ADMA, thus resolving a 20-year controversy and providing a starting point for the investigation of alternative, ADMA-independent functions of DDAH2.

Efficiency of Bone Marrow-Derived Mesenchymal Stem Cells and Hesperetin in the Treatment of Streptozotocin-Induced Type 1 Diabetes in Wistar Rats.

Type 1 diabetes mellitus (T1DM) was established to be ameliorated by islet transplantation, but the shortage of the transplanted human islet tissue and the use of immunosuppressive drugs to inhibit the rejection of allogeneic grafts make this type of therapy is limited. Nowadays, therapy with stem cells is one of the most promising future treatments. This kind of therapy could have a profound impact on both replacement, as well as regenerative therapies, to improve or even cure various disorders, including diabetes mellitus. Flavonoids have also been shown to possess anti-diabetic effects. Thus, this study aims to evaluate the effectiveness of the bone marrow-derived mesenchymal stem cells (BM-MSCs) and hesperetin in the treatment of a T1DM rat model. T1DM was induced in male Wistar rats that had been starved for 16 h via intraperitoneal injection of STZ at a dose of 40 mg/kg body weight (b.wt.). After 10 days of STZ injection, the diabetic rats were allocated into four groups. The first diabetic animal group was considered a diabetic control, while the other three diabetic animal groups were treated for six weeks, respectively, with hesperetin (given orally at a dose of 20 mg/kg b.wt.), BM-MSCs (injected intravenously at a dose of 1 × 106 cells/rat/week), and their combination (hesperetin and BM-MSCs). The use of hesperetin and BM-MSCs in the treatment of STZ-induced diabetic animals significantly improved the glycemic state, serum fructosamine, insulin and C-peptide levels, liver glycogen content, glycogen phosphorylase, glucose-6-phosphatase activities, hepatic oxidative stress, and mRNA expressions of NF-κB, IL-1β, IL-10, P53, and Bcl-2 in pancreatic tissue. The study suggested the therapy with both hesperetin and BM-MSCs produced marked antihyperglycemic effects, which may be mediated via their potencies to ameliorate pancreatic islet architecture and insulin secretory response, as well as to decrease hepatic glucose output in diabetic animals. The improvement effects of hesperetin and BM-MSCs on the pancreatic islets of diabetic rats may be mediated via their antioxidant, anti-inflammatory, and antiapoptotic actions.

Distinct Insulin Physiology Trajectories in Euglycemic Pregnancy and Gestational Diabetes Mellitus.

To evaluate changes in insulin physiology in euglycemic pregnancy and gestational diabetes mellitus (GDM). Participants underwent oral glucose tolerance tests at ≤15 weeks' gestation (early pregnancy), 24-32 weeks' gestation (mid-late pregnancy), and 6-24 weeks postpartum. We evaluated longitudinal changes in insulin secretory response (log Stumvoll first-phase estimate) and insulin sensitivity (log Matsuda index) using linear mixed models. We then evaluated participants who met GDM criteria in early pregnancy (early GDM) and mid-late pregnancy (classic GDM) separately from those without GDM. We derived the pregnancy insulin physiology (PIP) index to quantify β-cell compensation for insulin resistance. Among 166 participants, 21 had early GDM and 24 developed classic GDM. Insulin sensitivity was reduced slightly in early pregnancy (β = -0.20, P < 0.001) and substantially in mid-late pregnancy (β = -0.47, P < 0.001) compared with postpartum. Insulin secretory response (adjusted for insulin sensitivity) was augmented in early pregnancy (β = 0.16, P < 0.001) and mid-late pregnancy (β = 0.16, P = 0.001) compared with postpartum. Compared with postpartum, the PIP index was augmented in early pregnancy (β = 215, P = 0.04) but not mid-late pregnancy (β = 55, P = 0.64). Early GDM was distinguished by a substantial reduction in early pregnancy insulin sensitivity (β = -0.59, P < 0.001) compared with postpartum. Both early and classic GDM lacked evidence of early pregnancy augmentation of insulin secretory response (adjusted for insulin sensitivity) and the PIP index (P > 0.1 vs. postpartum). Early pregnancy PIP index predicted GDM independent of participant characteristics (area under the curve without PIP index 0.70 [95% CI 0.61-0.79], area under the curve with PIP index 0.87 [95% CI 0.80-0.93]). β-Cell function is enhanced in early pregnancy. Deficient first-trimester β-cell function predicts GDM.

Nutrient regulation of the islet epigenome controls adaptive insulin secretion.

Adaptation of the islet β cell insulin-secretory response to changing insulin demand is critical for blood glucose homeostasis, yet the mechanisms underlying this adaptation are unknown. Here, we have shown that nutrient-stimulated histone acetylation plays a key role in adapting insulin secretion through regulation of genes involved in β cell nutrient sensing and metabolism. Nutrient regulation of the epigenome occurred at sites occupied by the chromatin-modifying enzyme lysine-specific demethylase 1 (Lsd1) in islets. β Cell-specific deletion of Lsd1 led to insulin hypersecretion, aberrant expression of nutrient-response genes, and histone hyperacetylation. Islets from mice adapted to chronically increased insulin demand exhibited shared epigenetic and transcriptional changes. Moreover, we found that genetic variants associated with type 2 diabetes were enriched at LSD1-bound sites in human islets, suggesting that interpretation of nutrient signals is genetically determined and clinically relevant. Overall, these studies revealed that adaptive insulin secretion involves Lsd1-mediated coupling of nutrient state to regulation of the islet epigenome.

The differentiation and generation of glucose-sensitive beta like-cells from menstrual blood-derived stem cells using an optimized differentiation medium with platelet-rich plasma (PRP)

Regarding their reversible damage of insulin-producing cells (IPCs) and the inefficiency of treatment methods for type 1 diabetes mellitus (T1DM), scientists decided to produce IPCs from an unlimited source of cells. But the production of these cells is constantly faced with problems such as low differentiation efficiency in cell therapy and regenerative medicine. This study provided an ideal differentiation medium enriched with plasma-rich platelet (PRP) delivery to produce IPCs from menstrual blood-derived stem cells (MenSCs). We compared them with and without PRP differentiation medium. MenSCs were then cultured in two experimental groups: with/without PRP differentiation medium and a control group (undifferentiated MenSCs). After 18 days, differentiated cells were analyzed for expression of pancreatic gene markers by real-time PCR. Immunocytochemical staining was used to detect the presence of insulin and Pdx-1 in the differentiated cells, and insulin and C-peptide secretion response to glucose were tested by ELISA. Finally, the morphology of differentiated cells was examined by an inverted microscope. In vitro studies showed that MenSCs differentiated in the PRP differentiation medium had strong properties of IPCs such as pancreatic islet-like structure. The expression of pancreatic markers at both RNA and protein levels showed that the differentiation efficiency was higher in the PRP differentiation medium. In both experimental groups, the differentiated cells were functional and secreted C-peptide and insulin on glucose stimulation, but the secretion of C-peptide and insulin in the PRP group was higher than those cultured in the without PRP differentiation medium. Our findings showed that using of PRP enriched differentiation medium can promote the differentiation of MenSCs into IPCs compared to the without PRP culture group. Therefore, the use of PRP into differentiation media can be proposed as a new approach to producing IPCs from MenSCs and used in cell-based therapies for T1DM.