Β-cell Response Research Articles

Lower threshold to NFκB activity sensitizes murine β-cells to streptozotocin.

The β-cell response to injury may be as critical for the development of diabetes as the specific insult. In the current study, we used streptozotocin (STZ) to injure the β-cell in order to study the response with a focus on NFκB. MIN6 cells were exposed to STZ (0.5-8 mM, 0-24h) ±TNFα (100 ng/mL) and ±IκBβ siRNA to lower the threshold to NFκB activation. Cell viability was determined by trypan blue exclusion. NFκB activation was determined by the expression of the target genes Nos2 and Cxcl10, localization of the NFκB proteins p65 and p50, and expression and localization of the NFκB inhibitors, IκBβ and IκBα. There was no NFκB activation in MIN6 cell exposed to STZ (2 mM) alone. However, knocking down IκBβ expression using siRNA resulted in STZ-induced expression of NFκB target genes and increased cell death, while co-incubation with STZ and TNFα enhanced cell death compared to either exposure alone. Adult male IκBβ-/- and WT mice were exposed to STZ and monitored for diabetes. The IκBβ-/- mice developed hyperglycemia and diabetes more frequently than controls following STZ exposure. Based on these results we conclude that STZ exposure alone does not induce NFκB activity. However, lowering the threshold to NFκB activation by co-incubation with TNFα or lowering IκBβ levels by siRNA sensitizes the NFκB response to STZ and results in a higher likelihood of developing diabetes in vivo. Therefore, increasing the threshold to NFκB activation through stabilizing NFκB inhibitory proteins may prevent β-cell injury and the development of diabetes.

FXR-mediated epigenetic regulation of GLP-1R expression contributes to enhanced incretin effect in diabetes after RYGB.

In this study, we investigated how Roux-en-Y gastric bypass (RYGB) enhances glucagon-like peptide 1 (GLP-1) response in GK rats and explored the potential link between RYGB-stimulated BAs/FXR signalling and GLP-1R-linked signalling in β-cells, a key pathway that regulates glucose-stimulated insulin secretion (GSIS). Here we show that RYGB restores GLP-1R expression in GK rat islets. This involves increased total BAs as well as chenodeoxycholic acid (CDCA), leading to FXR activation, increasing FXR binding to the promoter of Glp-1r and enhancing occupancy of histone acetyltransferase steroid receptor coactivator-1 (SRC1), thus increasing histone H3 acetylation at the promoter. These coordinated events bring about increased GLP-1R expression, resulting in greater GLP-1 response in β-cells. Moreover, ablation ofFXR suppressed the stimulatory effects of GLP-1. Thus, this study unravels the crucial role of the BAs/FXR/SRC1 axis-controlled GLP-1R expression in β-cells, which results in enhanced incretin effect and normalized blood glucose of GK rats after RYGB.

Analysis of the behavior of 2D monolayers and 3D spheroid human pancreatic beta cells derived from induced pluripotent stem cells in a microfluidic environment

The limited availability of primary human β-cells/islets and their quality (due to donor diversity) restrict the development of in vitro models for diabetes research. Human induced pluripotent stem cells (hiPSCs) may be a promising cell-source for diabetes studies, anti-diabetic drug screening and personalized therapies. However, achieving levels of maturity/functionality that are comparable to the in vivo situation and islets rebuilt from iPSCs is still challenging. Here, we compare and discuss two strategies for culturing human pancreatic β-cells derived from hiPSCs in microfluidic biochips. First, we confirmed that the protocol in conventional Petri 2D monolayer led to insulin, PDX1 and MAFA positive staining, to C-Peptide productive cells, and to tissue responsive to high/low glucose and GLP1 stimulation. This protocol and its subsequent modifications (including extracellular matrix coating, cell adhesion time, cell inoculation density, flow rate) was not successful in the 2D biochip culture. We proposed a second strategy using 3D spheroids created from honeycomb static cultures. Spheroids in static experiments carried out over 14 days demonstrated that they expressed high levels of β-cell markers (INS mRNA) and higher α-cell markers (GCG mRNA and glucagon positive staining), when compared to Petri 2D cultures. Furthermore, the 3D spheroids were specifically able to secrete insulin in response to both high/low glucose stimulation and GLP1 exposure. The spheroids were successfully inoculated into biochips and maintained for 10 days in perfusion. The 3D biochip cultures increased mRNA levels of GCG and maintained high levels of β-cell markers and responsiveness to both high/low glucose and GLP1 stimulation. Finally, C-peptide and insulin secretion were higher in biochips when compared to static spheroids. These results illustrate the promising potential for hiPSCs derived β-cells and their spheroid-based pancreas-on-chip model for pancreatic disease/diabetes modeling and anti-diabetic drug screening.

Obstructive Sleep Apnea, Glucose Tolerance, and β-Cell Function in Adults With Prediabetes or Untreated Type 2 Diabetes in the Restoring Insulin Secretion (RISE) Study

Obstructive sleep apnea (OSA) is associated with insulin resistance and has been described as a risk factor for type 2 diabetes. Whether OSA adversely impacts pancreatic islet β-cell function remains unclear. We aimed to investigate the association of OSA and short sleep duration with β-cell function in overweight/obese adults with prediabetes or recently diagnosed, treatment-naive type 2 diabetes. Two hundred twenty-one adults (57.5% men, age 54.5 ± 8.7 years, BMI 35.1 ± 5.5 kg/m2) completed 1 week of wrist actigraphy and 1 night of polysomnography before undergoing a 3-h oral glucose tolerance test (OGTT) and a two-step hyperglycemic clamp. Associations of measures of OSA and actigraphy-derived sleep duration with HbA1c, OGTT-derived outcomes, and clamp-derived outcomes were evaluated with adjusted regression models. Mean ± SD objective sleep duration by actigraphy was 6.6 ± 1.0 h/night. OSA, defined as an apnea-hypopnea index (AHI) of five or more events per hour, was present in 89% of the participants (20% mild, 28% moderate, 41% severe). Higher AHI was associated with higher HbA1c (P = 0.007). However, OSA severity, measured either by AHI as a continuous variable or by categories of OSA severity, and sleep duration (continuous or <6 vs. ≥6 h) were not associated with fasting glucose, 2-h glucose, insulin sensitivity, or β-cell responses. In this baseline cross-sectional analysis of the RISE clinical trial of adults with prediabetes or recently diagnosed, untreated type 2 diabetes, the prevalence of OSA was high. Although some measures of OSA severity were associated with HbA1c, OSA severity and sleep duration were not associated with measures of insulin sensitivity or β-cell responses.

Lettuce-avicularin treatment reverses insulin resistance through stimulation of glycolytic kinases and insulin signaling molecules.

Objective(s):In order to recommend a more effective approach to manage insulin resistance, we monitored the activities of glycolytic kinases, insulin signaling molecules, and incretin hormones and identified the possible targets related to the insulin-sensitizing effects of combined pharmacological and dietary intervention involving avicularin and lettuce.Materials and Methods:Insulin resistance was induced in rats with a fructose-rich diet and confirmed from baseline analysis of FBS (>250 mg/dl), insulin (>25 µIU/ml), and HOMA-IR (>10). For 12 weeks, the insulin-resistant rats were treated exclusively with 5000 mg/kg b.w avicularin (DAvi) or by dietary placement on lettuce (DLet) or a combination of both and compared with non-insulin resistant rats.Results:Avicularin reversed alterations in HbA1c and insulin levels. DLet produced no significant effect on the incretins GLP 1 (P=0.909) and GIP (P=0.990), but DAvi slightly stimulated GLP 1 but not GIP. A strong positive correlation was found between improved β-cell responsiveness and the insulin signaling molecules: Akt2 (r=0.7248), IRS 1 (r=0.5173), and PI3K (r=0.7892). Only the combined avicularin and lettuce reversed the Akt2 levels (P=0.728). The lettuce meal slightly stimulated PI3K but normalized IRS 1 while avicularin treatment slightly stimulated IRS 1 but restored the PI3K levels (P=0.815). The positive correlation between β-cell responsiveness and hexokinase (r=0.5959), PFK (r=0.6222), and PK (r=0.6591) activities were statistically significant. Alterations in glycolytic kinases were reversed by DLet and in combination with avicularin.Conclusion:A combined pharmacological and dietary approach with avicularin and lettuce is required to effectively reverse insulin resistance.

Glucose Metabolism and Pyruvate Carboxylase Enhanceglutathione Synthesis and Restrict Oxidative Stress in Beta-Cells

Glucose metabolism modulates β-cell responses to diabetogenic stress, including inflammation. Here, we probed the specific metabolic mechanisms that relay the protective effect of glucose in beta-cell inflammation by interrogating the metabolite profiles of primary islets from human donors and identified de novo glutathione synthesis as a prominent glucose-driven pro-survival pathway. We find that pyruvate carboxylase is required for glutathione synthesis in β-cells and promotes their antioxidant capacity to counter inflammation and nitrosative stress. Loss- and gain-of-function studies indicate that pyruvate carboxylase is necessary and sufficient to mediate the metabolic input from glucose into glutathione synthesis and the oxidative stress response. Altered redox metabolism and cellular capacity to replenish glutathione pools are relevant in multiple pathologies beyond obesity and diabetes. Our findings reveal a direct interplay between glucose metabolism and glutathione biosynthesis via pyruvate carboxylase. This metabolic axis may also have implications in other settings where sustaining glutathione pools is essential.

Mechanisms controlling pancreatic islet cell function in insulin secretion.

Metabolic homeostasis in mammals is tightly regulated by the complementary actions of insulin and glucagon. The secretion of these hormones from pancreatic β-cells and α-cells, respectively, is controlled by metabolic, endocrine, and paracrine regulatory mechanisms and is essential for the control of blood levels of glucose. The deregulation of these mechanisms leads to various pathologies, most notably type 2 diabetes, which is driven by the combined lesions of impaired insulin action and a loss of the normal insulin secretion response to glucose. Glucose stimulates insulin secretion from β-cells in a bi-modal fashion, and new insights about the underlying mechanisms, particularly relating to the second or amplifying phase of this secretory response, have been recently gained. Other recent work highlights the importance of α-cell-produced proglucagon-derived peptides, incretin hormones from the gastrointestinal tract and other dietary components, including certain amino acids and fatty acids, in priming and potentiation of the β-cell glucose response. These advances provide a new perspective for the understanding of the β-cell failure that triggers type 2 diabetes.

Limitations of the fasting proinsulin to insulin ratio as a measure of β-cell health in people with and without impaired glucose tolerance.

The fasting proinsulin to insulin ratio is elevated in people with type 2 diabetes and has been suggested as a marker of β-cell health. However, its utility in discriminating between individuals with varying degrees of β-cell dysfunction is unclear. Proinsulin has a very different half-life to insulin and unlike insulin does not undergo hepatic extraction prior to reaching the systemic circulation. Given these limitations, we sought to examine the relationship between fasting and postprandial concentrations of β-cell polypeptides (proinsulin, insulin and C-peptide) in people with normal and impaired glucose tolerance in differing metabolic environments. Subjects were studied on two occasions in random order while undergoing an oral challenge. During one study day, free fatty acids were elevated (to induce insulin resistance) by infusion of Intralipid with heparin. Proinsulin to insulin and proinsulin to C-peptide ratios were calculated for the 0-, 30-, 60- and 240-minute time points. Insulin action (Si) and β-cell responsivity (Φ) indices were calculated using the oral minimal model. The fasting proinsulin to c-peptide or fasting proinsulin to insulin ratios did not differ between groups and did not predict subsequent β-cell responsivity to glucose during the glycerol or Intralipid study days in either group. Among nondiabetic individuals, the fasting proinsulin to insulin ratio is not a useful marker of β-cell function.

Inhibition of mitochondrial oxidative metabolism attenuates EMCV replication and protects β-cells from virally mediated lysis

Viral infection is one environmental factor that may contribute to the initiation of pancreatic β-cell destruction during the development of autoimmune diabetes. Picornaviruses, such as encephalomyocarditis virus (EMCV), induce a pro-inflammatory response in islets leading to local production of cytokines, such as IL-1, by resident islet leukocytes. Furthermore, IL-1 is known to stimulate β-cell expression of iNOS and production of the free radical nitric oxide. The purpose of this study was to determine whether nitric oxide contributes to the β-cell response to viral infection. We show that nitric oxide protects β-cells against virally mediated lysis by limiting EMCV replication. This protection requires low micromolar, or iNOS-derived, levels of nitric oxide. At these concentrations nitric oxide inhibits the Krebs enzyme aconitase and complex IV of the electron transport chain. Like nitric oxide, pharmacological inhibition of mitochondrial oxidative metabolism attenuates EMCV-mediated β-cell lysis by inhibiting viral replication. These findings provide novel evidence that cytokine signaling in β-cells functions to limit viral replication and subsequent β-cell lysis by attenuating mitochondrial oxidative metabolism in a nitric oxide–dependent manner.

Collagen and Endothelial Cell Coculture Improves β-Cell Functionality and Rescues Pancreatic Extracellular Matrix.

The use of biomaterials and biomaterial functionalization is a promising approach to support pancreatic islet viability posttransplantation in an effort to reduce insulin dependence for patients afflicted with diabetes mellitus type 1. Extracellular matrix (ECM) proteins are known to impact numerous reparative functions in the body. Assessing how endogenously expressed pancreatic ECM proteins are affected by posttransplant-like hypoxic conditions may provide significant insights toward the development of tissue-engineered therapeutic strategies to positively influence β-cell survival, proliferation, and functionality. Here, we investigated the expression of three relevant groups of pancreatic ECM proteins in human native tissue, including basement membrane (BM) proteins (collagen type 4 [COL4], laminins [LAM]), proteoglycans (decorin [DCN], nidogen-1 [NID1]), and fibril-forming proteins (fibronectin [FN], collagen type 1 [COL1]). In an in vitro hypoxia model, we identified that ECM proteins were differently affected by hypoxic conditions, contributing to an overall loss of β-cell functionality. The use of a COL1 hydrogel as carrier material demonstrated a protective effect on β-cells mitigating the effect of hypoxia on proteoglycans as well as fibril-forming protein expression, supporting β-cell functionality in hypoxia. We further showed that providing endothelial cells (ECs) into the COL1 hydrogel improves β-cell response as well as the expression of relevant BM proteins. Our data show that β-cells benefit from a microenvironment composed of structure-providing COL1 with the incorporation of ECs to withstand the harsh conditions of hypoxia. Such hydrogels support β-cell survival and can serve as an initial source of ECM proteins to allow cell engraftment while preserving cell functionality posttransplantation. Impact statement Expression analysis identifies hypoxia-induced pathological changes in extracellular matrix (ECM) homeostasis as potential targets to support β-cell transplants by encapsulation in biomaterials for the treatment of diabetes mellitus. A collagen-1 hydrogel is shown to attenuate the effect of hypoxia on β-cells and their ECM expression. The functionalization of the hydrogel with endothelial cells increases the β-cell response to glucose and rescues essential basement membrane proteins.

Potentiation of incretin hormones and modulation of metabolic enzymes as possible mechanisms behind the insulin sensitizing effects of cabbage-metformin treatment

In our study, we treated high fructose diet-induced insulin resistance in rats with any of metformin, cabbage (80%w/w) or combined metformin and cabbage (MetCabb), and observed the activities of glycolysis and gluconeogenesis regulatory enzymes, incretin hormones and other hormones affecting glucose homeostasis. Comparisons were made with normoglycemic noninsulin resistance rats (control) and insulin-resistant untreated rats (INres). Baseline analysis showing elevated fasting blood sugar (>250 mg/dl), insulin (>25 µIU/ml) and HOMA-IR (>10) satisfied the criteria for recruitment into the insulin-resistant groups. Treatment lasted for 12 weeks. HOMA-IR values significantly (P < 0.05) decreased from 24.7 to 5.5 and 10.6 respectively with MetCabb treatment. MetCabb normalized HOMA-IR values and mean β-cell responsiveness of the INres. Cabbage and metCabb normalized the leptin levels relative to control. The mean fasting blood sugar, insulin, and c-peptide levels with MetCabb treatment reverted to control levels. We found a strong positive linear correlation between the glucagon levels (r = 0.9145) and increasing HOMA-IR values while both incretin hormones; GLP-1 and GIP negatively regressed (r = -0.8084 and -0.8488). MetCab treatment produced comparable values of GLP-1 and GIP to the control. A strong positive correlation was found between the HOMA-IR values and the PEPCK (r = 0.9065), F-1,6-BPase (r = 0.7951), and G-6-Pase (r = 0.7893). The hexokinase (r = -0.807), PFK (r = -0.9151), and PK (r = -0.7448) levels regressed as HOMA-IR values increased. The glycolytic and gluconeogenic enzymes except PEPCK reverted to control levels with MetCabb treatment. Combination of metformin and cabbage was more effective than individual treatments.

Withdrawal of medications leads to worsening of OGTT parameters in youth with impaired glucose tolerance or recently-diagnosed type 2 diabetes.

The RISE Pediatric Medication Study compared strategies for preserving β-cell function, including a 9-month follow-up after treatment withdrawal to test treatment effect durability. Evaluate OGTT measures of glucose and β-cell response through 12 months of intervention and 9 months of medication washout. Youth (n = 91) aged 10 to 19 years with BMI ≥85th percentile and impaired glucose tolerance (IGT) or recently diagnosed type 2 diabetes (T2D). A multicenter randomized clinical trial comparing insulin glargine for 3 months followed by metformin for 9 months (G→Met) or metformin alone (Met) for 12 months. We report within-group changes from baseline to end of medication intervention (M12), baseline to 9 months post-medication withdrawal (M21), and end of medication (M12) to M21. OGTT C-peptide index [CPI] paired with 1/fasting insulin evaluated β-cell response. At M12, both treatments were associated with stable fasting glucose (G→Met baseline 6.0 ± 0.1 vs M12 5.9 ± 0.2 mmol/L, P = .62; Met baseline 6.1 ± 0.2 vs M12 6.0 ± 0.2 mmol/L, P = .73) and 2-hour glucose (G→Met baseline 10.2 ± 0.4 vs M12 9.3 ± 0.5 mmol/L, P = .03; Met baseline 10.2 ± 0.4 vs M12 10.6 ± 0.6 mmol/L, P = .88). Following medication withdrawal, fasting glucose worsened (G→Met M21 8.6 ± 1.8, P = .004; Met M21 7.8 ± 0.7 mmol/L, P = .003), as did 2-hour glucose (G→Met M21 13.2 ± 1.4, P = .002; Met M21 13.1 ± 1.2 mmol/L, P = .006), associated with declines in β-cell response. G→Met and Met were associated with stable glucose measures during 12 months of treatment in youth with IGT or recently diagnosed T2D. Glucose and β-cell response worsened post-medication withdrawal, suggesting treatment must be long-term or alternative treatments pursued.

The KINGS Ins2 +/G32S Mouse: A Novel Model of β-Cell Endoplasmic Reticulum Stress and Human Diabetes.

Animal models are important tools in diabetes research because ethical and logistical constraints limit access to human tissue. β-Cell dysfunction is a common contributor to the pathogenesis of most types of diabetes. Spontaneous hyperglycemia was developed in a colony of C57BL/6J mice at King’s College London (KCL). Sequencing identified a mutation in the Ins2 gene, causing a glycine-to-serine substitution at position 32 on the B chain of the preproinsulin 2 molecule. Mice with the Ins2+/G32S mutation were named KCL Ins2 G32S (KINGS) mice. The same mutation in humans (rs80356664) causes dominantly inherited neonatal diabetes. Mice were characterized, and β-cell function was investigated. Male mice became overtly diabetic at ∼5 weeks of age, whereas female mice had only slightly elevated nonfasting glycemia. Islets showed decreased insulin content and impaired glucose-induced insulin secretion, which was more severe in males. Transmission electron microscopy and studies of gene and protein expression showed β-cell endoplasmic reticulum (ER) stress in both sexes. Despite this, β-cell numbers were only slightly reduced in older animals. In conclusion, the KINGS mouse is a novel model of a human form of diabetes that may be useful to study β-cell responses to ER stress.

Hypoxia-inducible factor-1 mediates pancreatic β-cell dysfunction by intermittent hypoxia.

The role of hypoxia-inducible factor (HIF)-1 in pancreatic β-cell response to intermittent hypoxia (IH) was examined. Studies were performed on adult wild-type (WT), HIF-1α heterozygous (HET), β-cell-specific HIF-1-/- mice and mouse insulinoma (MIN6) cells exposed to IH patterned after blood O2 profiles during obstructive sleep apnea. WT mice treated with IH showed insulin resistance, and pancreatic β-cell dysfunction manifested as augmented basal insulin secretion, and impaired glucose-stimulated insulin secretion and these effects were absent in HIF-1α HET mice. IH increased HIF-1α expression and elevated reactive oxygen species (ROS) levels in β-cells of WT mice. The elevated ROS levels were due to transcriptional upregulation of NADPH oxidase (NOX)-4 mRNA, protein and enzymatic activity, and these responses were absent in HIF-1α HET mice as well as in β-HIF-1-/- mice. IH-evoked β-cell responses were absent in adult WT mice treated with digoxin, an inhibitor of HIF-1α. MIN6 cells treated with in vitro IH showed enhanced basal insulin release and elevated HIF-1α protein expression, and these effects were abolished with genetic silencing of HIF-1α. IH increased NOX4 mRNA, protein, and enzyme activity in MIN6 cells and disruption of NOX4 function by siRNA or scavenging H2O2 with polyethylene glycol catalase blocked IH-evoked enhanced basal insulin secretion. These results demonstrate that HIF-1-mediated transcriptional activation of NOX4 and the ensuing increase in H2O2 contribute to IH-induced pancreatic β-cell dysfunction.

Combined transcriptome and proteome profiling of the pancreatic \u03b2-cell response to palmitate unveils key pathways of \u03b2-cell lipotoxicity

BackgroundProlonged exposure to elevated free fatty acids induces β-cell failure (lipotoxicity) and contributes to the pathogenesis of type 2 diabetes. In vitro exposure of β-cells to the saturated free fatty acid palmitate is a valuable model of lipotoxicity, reproducing features of β-cell failure observed in type 2 diabetes. In order to map the β-cell response to lipotoxicity, we combined RNA-sequencing of palmitate-treated human islets with iTRAQ proteomics of insulin-secreting INS-1E cells following a time course exposure to palmitate.ResultsCrossing transcriptome and proteome of palmitate-treated β-cells revealed 85 upregulated and 122 downregulated genes at both transcript and protein level. Pathway analysis identified lipid metabolism, oxidative stress, amino-acid metabolism and cell cycle pathways among the most enriched palmitate-modified pathways. Palmitate induced gene expression changes compatible with increased free fatty acid mitochondrial import and β-oxidation, decreased lipogenesis and modified cholesterol transport. Palmitate modified genes regulating endoplasmic reticulum (ER) function, ER-to-Golgi transport and ER stress pathways. Furthermore, palmitate modulated cAMP/protein kinase A (PKA) signaling, inhibiting expression of PKA anchoring proteins and downregulating the GLP-1 receptor. SLC7 family amino-acid transporters were upregulated in response to palmitate but this induction did not contribute to β-cell demise. To unravel critical mediators of lipotoxicity upstream of the palmitate-modified genes, we identified overrepresented transcription factor binding sites and performed network inference analysis. These identified LXR, PPARα, FOXO1 and BACH1 as key transcription factors orchestrating the metabolic and oxidative stress responses to palmitate.ConclusionsThis is the first study to combine transcriptomic and sensitive time course proteomic profiling of palmitate-exposed β-cells. Our results provide comprehensive insight into gene and protein expression changes, corroborating and expanding beyond previous findings. The identification of critical drivers and pathways of the β-cell lipotoxic response points to novel therapeutic targets for type 2 diabetes.

Short-term inhibition of autophagy benefits pancreatic β-cells by augmenting ether lipids and peroxisomal function, and by countering depletion of n-3 polyunsaturated fatty acids after fat-feeding

ObjectiveInvestigations of autophagy in β-cells have usually focused on its homeostatic function. More dynamic roles in inhibiting glucose-stimulated insulin secretion (GSIS), potentially involving remodelling of cellular lipids, have been suggested from in vitro studies but not evaluated in vivo. MethodsWe employed temporally-regulated deletion of the essential autophagy gene, Atg7, in β-cells. Mice were fed chow or high-fat diets (HFD), in conjunction with deletion of Atg7 for the last 3 weeks (short-term model) or 9 weeks (long-term model). Standard in vivo metabolic phenotyping was undertaken, and 450 lipid species in islets quantified ex vivo using mass spectroscopy (MS). MIN6 cells were also employed for lipidomics and secretory interventions. Resultsβ-cell function was impaired by inhibiting autophagy in the longer-term, but conversely improved by 3-week deletion of Atg7, specifically under HFD conditions. This was accompanied by augmented GSIS ex vivo. Surprisingly, the HFD had minimal effect on sphingolipid and neutral lipid species, but modulated >100 phospholipids and ether lipids, and markedly shifted the profile of polyunsaturated fatty acid (PUFA) sidechains from n3 to n6 forms. These changes were partially countered by Atg7 deletion, consistent with an accompanying upregulation of the PUFA elongase enzyme, Elovl5. Loss of Atg7 separately augmented plasmalogens and alkyl lipids, in association with increased expression of Lonp2, a peroxisomal chaperone/protease that facilitates maturation of ether lipid synthetic enzymes. Depletion of PUFAs and ether lipids was also observed in MIN6 cells chronically exposed to oleate (more so than palmitate). GSIS was inhibited by knocking down Dhrs7b, which encodes an enzyme of peroxisomal ether lipid synthesis. Conversely, impaired GSIS due to oleate pre-treatment was selectively reverted by Dhrs7b overexpression. ConclusionsA detrimental increase in n6:n3 PUFA ratios in ether lipids and phospholipids is revealed as a major response of β-cells to high-fat feeding. This is partially reversed by short-term inhibition of autophagy, which results in compensatory changes in peroxisomal lipid metabolism. The short-term phenotype is linked to improved GSIS, in contrast to the impairment seen with the longer-term inhibition of autophagy. The balance between these positive and negative inputs could help determine whether β-cells adapt or fail in response to obesity.

1792-P: Measures of ß-Cell Function from Two-Step Hyperglycemic Clamps: Relation to Glycemia

We derived β-cell measures compensating for insulin resistance (disposition indices [DI]) from two-step hyperglycemic clamps with arginine stimulation, and examined their associations with glycemia cross-sectionally and longitudinally after treatment. Data included 355 adults (age 23-66 y; 51% female) and 89 youth (age 10-19 y; 71% female) from the RISE Consortium studies. Clamps were done at baseline and after 12 mo treatment with medications or gastric banding. Insulin sensitivity was quanitfied as M/I. β-cell responses included acute insulin (AIRg) and C-peptide (ACPRg) responses to glucose; steady-state C-peptide (SSCP) at glucose ∼11.1 mmol/L; and maximum insulin (AIRmax) or C-peptide (ACPRmax) responses to arginine at glucose &amp;gt;25 mmol/L. DIs were calculated from M/I and β-cell responses as [responses x (M/I)-b ]. The Table shows the correlations between each DI and glucose at baseline, and changes after treatment. The correlations were higher in youth than adults, except for changes in AIRmax or ACPRmax and 2-hour glucose. The correlations for ACPRg and AIRg were generally higher, followed by SSCP and then maximal responses. Thus, in the RISE cohorts, measures of early β-cell response were more strongly associated with glycemia than were late responses, with stronger correlations in youth. This underscores different contributions of early and late insulin secretion in the regulation of glycemia. Disclosure A. Xiang: None. T.S. Hannon: None. K.J. Mather: Research Support; Self; Abbott, Merck &amp; Co., Inc., Novo Nordisk Inc., Sanofi. E. Barengolts: None. M. Cree-Green: Advisory Panel; Self; Novo Nordisk Inc. K.J. Nadeau: None. S. Sam: None. S.E. Kahn: Advisory Panel; Self; Boehringer Ingelheim International GmbH, Eli Lilly and Company, Intarcia Therapeutics, Janssen Scientific Affairs, LLC., Merck &amp; Co., Inc., Novo Nordisk A/S, Pfizer Inc. T.A. Buchanan: None. T. Consortium: None. Funding American Diabetes Association (1-14-RISE-01 to S.E.K.); National Institute of Diabetes and Digestive and Kidney Diseases

1268-P: Associations between ß-Cell Function and Cognitive Measures following Treatment in Youth with Impaired Glucose Tolerance (IGT) or Recently Diagnosed Type 2 Diabetes (T2D)

In cross-sectional studies, adolescents with T2D show poorer cognition than normoglycemic youth. In the Restoring Insulin Secretion (RISE) Study, we assessed longitudinal relationships between insulin sensitivity, β-cell function and cognition in 72 youth (mean age 14.3±2.0, 10-18 y) with IGT or recently diagnosed T2D, randomized to 12 months of metformin (MET) or 3 months of glargine followed by 9 months of metformin (G/M). Hyperglycemic clamps were used to measure insulin sensitivity and β-cell responses: acute (0-10 min) C-peptide response to glucose, steady-state C-peptide (SSCP) at glucose of 11.1 mmol/L, and arginine-stimulated maximum C-peptide response (ACPRmax) at glucose &amp;gt;25 mmol/L. A cognitive battery (CogStateTM and story recall) assessed 1) reaction time; 2) visual discrimination; 3) attention and working memory; and 4) learning and episodic memory. A composite measure of cognition was constructed by scaling the sum of the four domain scores. Linear regression models were fit to assess relationships between cognition, treatment and β-cell function. Treatment group models were adjusted for baseline cognitive function, HbA1c and fasting glucose. β-cell models were adjusted for baseline cognitive and β-cell function. Cognitive composite scores improved over the 12-month treatment period (p&amp;lt;0.001), with no differences between treatment arms. Greater baseline ACPRmax predicted greater improvement in composite scores over 12 months in the G/M (p&amp;lt;0.05), but not MET group (p=0.61). Change in SSCP over the 12-month treatment was positively associated with increased composite scores in the G/M (p&amp;lt;0.05) but not MET group (p=0.35). Thus, improved cognition over the 12-month treatment with G/M in adolescents with IGT or early T2D may be related to practice effects with repeated testing, to changes in β-cell function or to other impacts of insulin treatment. Disclosure S. Craft: Advisory Panel; Self; vTv Therapeutics. M. Tripputi: None. S. Edelstein: None. M. Espeland: Other Relationship; Self; Boehringer Ingelheim International GmbH, Ironwood Pharmaceuticals. A. Claxton: Employee; Self; Alkermes plc. S.A. Arslanian: Research Support; Self; National Institutes of Health. Other Relationship; Self; AstraZeneca, Eli Lilly and Company, Novo Nordisk Inc. E. Barengolts: None. T.S. Hannon: None. S.E. Kahn: Advisory Panel; Self; Boehringer Ingelheim International GmbH, Eli Lilly and Company, Intarcia Therapeutics, Janssen Scientific Affairs, LLC., Merck &amp; Co., Inc., Novo Nordisk A/S, Pfizer Inc. K.J. Mather: Research Support; Self; Abbott, Merck &amp; Co., Inc., Novo Nordisk Inc., Sanofi. B. Mokhlesi: None. K.J. Nadeau: None. A. Sanderlin: None. K.A. Temple: None. P. Zeitler: Consultant; Self; Boehringer Ingelheim Pharmaceuticals, Inc., Daiichi Sankyo, Eli Lilly and Company, Merck &amp; Co., Inc. T. Consortium: None. Funding American Diabetes Association (1-14-RISE-01 to S.E.K.); National Institute of Diabetes and Digestive and Kidney Diseases

54-OR: Nitric Oxide Attenuates Viral Replication in Pancreatic ß-Cells through an Inhibition of Mitochondrial Oxidative Metabolism

Viral infection has been identified as an environmental factor that may play a role in initiating the destruction of pancreatic β-cells during the development of autoimmune diabetes. We have identified a pro-inflammatory response activated in pancreatic islets in response to infection with encephalomyocarditis virus (EMCV). In response to the local production of cytokines such as IL-1 by resident islet macrophages, β-cells produce nitric oxide, a free radical with known antiviral activity. Nitric oxide has been characterized both as a potent antiviral molecule and as a mediator of β-cell dysfunction and damage and so we investigated the role of nitric oxide in controlling β-cell viability in the context of EMCV infection. We find that nitric oxide, either supplied exogenously with donor compounds or produced endogenously in response to cytokine treatment, attenuates EMCV replication and protects β-cells against EMCV-mediated death. These protective actions of nitric oxide are restricted to conditions in which nitric oxide is produced at low micromolar levels (consistent with the levels of nitric oxide generated by iNOS) and are associated with the ability of nitric oxide to impair mitochondrial oxidation and decrease ATP levels in β-cells. Treatment with rotenone, an inhibitor of complex I of the electron transport chain, likewise attenuates EMCV replication in β-cells, which are reliant on oxidative metabolism for ATP generation, but not in fibroblasts, which can sustain ATP levels via glycolysis alone. These findings suggest a mechanism of protection restricted to β-cells where nitric oxide production attenuates viral replication by inhibiting oxidative metabolism and depleting cellular stores of ATP, supporting a protective role for nitric oxide in the response of β-cells to viral infection. Disclosure J. Stafford: None. J.A. Corbett: None. Funding American Heart Association (17PRE3253000); National Institutes of Health (DK052194, AI44458)

2117-P: BET Bromodomain Inhibitors Mitigate Cytokine-Induced Transcription in ß-Cells via Inhibition of Nf-κB

Type 1 diabetes is an autoimmune disease characterized by chronic inflammation of pancreatic islets, mediated by proinflammatory cytokines, leading to reduced response to glucose and ultimately selective destruction of insulin-producing β-cells. In common with many autoimmune diseases, the exact causes are poorly understood. Although genetic predisposition is a factor, low concordance rates of autoimmune diabetes among monozygotic twins suggest an, as yet undetermined, contribution of epigenetic factors. Recent studies have begun to explore the contributions of epigenetic regulatory proteins in onset and progression of autoimmune diabetes. An important class of epigenetic “readers” is the bromodomain and extraterminal domain (BET) family (Brd2, Brd3, Brd4, and Brdt), which has emerged as promising drug targets for a wide variety of diseases. A unique trait of the BET family is that these proteins contain two bromodomains (BD1 and BD2) that afford them the capacity to simultaneously bind two acetylated histones and/or non-histone proteins such as DNA-binding factors to modulate transcription in a cell-type specific manner. Here, we use an insulinoma β-cell line (INS832/13) and primary rat islets to investigate the ability of chemical inhibitors of BET bromodomain proteins to decrease the β-cell response to proinflammatory cytokines. We found that BET inhibitors mitigated cytokine-induced transcription of inflammatory mediators known to damage β-cells. This is mediated through the inhibition of the NF-κB pathway by BET inhibitors. Furthermore, using CRISPR/Cas9 knockout insulinoma β-cell lines of Brd2, Brd3, and Brd4 and BD1 vs. BD2 specific chemical inhibitors, we identified key mechanisms through which this altered transcriptional profile occurs. This work provides important mechanistic insights to disease onset and progression of autoimmune diabetes for which current therapies are ineffective. Disclosure J. Nord: None. S. Wynia-Smith: None. B.C. Smith: None. Funding American Diabetes Association (1-18-IBS-068 to B.C.S.); National Institute of Diabetes and Digestive and Kidney Diseases (R01DK119359)