Stress In Pancreatic Β-cells Research Articles

Clinacanthus nutans leaf extract reduces pancreatic β-cell apoptosis by inhibiting JNK activation and modulating oxidative stress and inflammation in streptozotocin-induced diabetic rats.

Controlling apoptosis induced by oxidative stress in pancreatic β-cells provides promising strategies for preventing and treating diabetes. Clinacanthus nutans leaves possess bioactive constituents with potential antioxidant and anti-diabetic properties. This study aimed to investigate the molecular mechanisms by which C. nutans extract protects pancreatic β-cells from apoptotic damage in streptozotocin (STZ)-induced diabetic rats. Diabetes was induced in male Wistar rats by intraperitoneal injection of 45 mg/kg STZ, followed by 28 days of treatment with C. nutans leaf extract and Glibenclamide as the standard drug. At the end of the study, blood samples were collected to measure glucose levels, oxidative stress markers, and inflammation. Pancreatic tissue was stained immunohistochemically to detect c-Jun N-terminal kinase (JNK) and Caspase-3 expression. The administration of C. nutans leaf extract to diabetic rats significantly reduced fasting blood glucose, malondialdehyde, and tumor necrosis factor-α levels, while concurrently enhancing the activity of superoxide dismutase. The immunohistochemical studies revealed a decrease in the expression of JNK and caspase-3 in the pancreatic islets of diabetic rats. Clinacanthus nutans exhibits the potential to protect pancreatic β-cells from apoptosis by suppressing oxidative stress and inflammation.

CDN1163, an activator of sarco/endoplasmic reticulum Ca2+ ATPase, up-regulates mitochondrial functions and protects against lipotoxicity in pancreatic β-cells.

High levels of Ca2+ in the endoplasmic reticulum (ER), established by the sarco/endoplasmic reticulum Ca2+ ATPase (SERCA), are required for protein folding and cell signalling. Excessive ER Ca2+ release or decreased SERCA activity induces unfolded protein accumulation and ER stress in pancreatic β-cells, leading to defective insulin secretion and diabetes. Here we have investigated the consequences of enhancing ER Ca2+ uptake on β-cell survival and function. The effects of SERCA activator, CDN1163, on Ca2+ homeostasis, protein expression, mitochondrial activities, insulin secretion, and lipotoxicity have been studied in mouse pancreatic β-cells and MIN6 cells. CDN1163, increased insulin synthesis and exocytosis from islets. CDN1163 also increased the sensitivity of the cytosolic Ca2+ oscillation response to glucose and potentiated it in dispersed and sorted β-cells. CDN1163 augmented the ER and mitochondrial Ca2+ content, the mitochondrial membrane potential, respiration, and ATP synthesis. CDN1163 up-regulated expression of inositol 1,4,5-trisphosphate receptors and antioxidant enzymes, and mitochondrial biogenesis, including peroxisome proliferator-activated receptor γ coactivator 1α (PGC1α). Overexpression of SERCA2a or 2b replicated the effects of CDN1163, while knockdown of SERCA2 abolished the stimulatory actions of CDN1163. In palmitate-treated β-cells, CDN1163 prevented ER Ca2+ depletion, mitochondrial dysfunction, cytosolic and mitochondrial oxidative stress, defective insulin secretion, and apoptotic cell death. Activation of SERCA enhanced mitochondrial bioenergetics and antioxidant capability, suppressing the cytotoxic effects of palmitate. Our results suggest that targeting SERCA could be a novel therapeutic strategy to protect β-cells from lipotoxicity and the development of Type 2 diabetes.

Protective Effect of Hawthorn Fruit Extract against High Fructose-Induced Oxidative Stress and Endoplasmic Reticulum Stress in Pancreatic β-Cells

Hyperglycemia has deleterious effects on pancreatic β-cells, causing dysfunction and insulin resistance that lead to diabetes mellitus (DM). The possible causes of injury can be caused by glucose- or fructose-induced oxidative and endoplasmic reticulum (ER) stress. Hawthorn (Crataegus pinnatifida) fruit has been widely used as a hypolipidemic agent in traditional herbal medicine. The study aimed to investigate whether high fructose-induced pancreatic β-cell dysfunction could be reversed through amelioration of ER stress by the treatment of polyphenol-enriched extract (PEHE) from hawthorn fruit. The extract was partitioned using ethyl acetate as a solvent from crude water extract (WE) of hawthorn fruits, followed by column fractionation. The results showed that the contents of total polyphenols, flavonoids and triterpenoids in PEHE could be enhanced by 2.2-, 7.7- and 1.1-fold, respectively, in comparison to the original obtained WE from hawthorn fruit. In ER stress studies, a sharp increase in the inhibitory activity on the gene expression levels of GRP79, ATF6, IRE1α and CHOP involved in ER stress was evident when dosages of PEHE at 50-100 μg/mL were used against high-fructose (150 mM)-treated cells. HPLC-MS/MS analysis showed that polyphenols and flavonoids collectively accounted for 87.03% of the total content of PEHE.

The Role of ER Stress in Diabetes: Exploring Pathological Mechanisms Using Wolfram Syndrome.

The endoplasmic reticulum (ER) is a cytosolic organelle that plays an essential role in the folding and processing of new secretory proteins, including insulin. The pathogenesis of diabetes, a group of metabolic disorders caused by dysfunctional insulin secretion (Type 1 diabetes, T1DM) or insulin sensitivity (Type 2 diabetes, T2DM), is known to involve the excess accumulation of "poorly folded proteins", namely, the induction of pathogenic ER stress in pancreatic β-cells. ER stress is known to contribute to the dysfunction of the insulin-producing pancreatic β-cells. T1DM and T2DM are multifactorial diseases, especially T2DM; both environmental and genetic factors are involved in their pathogenesis, making it difficult to create experimental disease models. In recent years, however, the development of induced pluripotent stem cells (iPSCs) and other regenerative technologies has greatly expanded research capabilities, leading to the development of new candidate therapies. In this review, we will discuss the mechanism by which dysregulated ER stress responses contribute to T2DM pathogenesis. Moreover, we describe new treatment methods targeting protein folding and ER stress pathways with a particular focus on pivotal studies of Wolfram syndrome, a monogenic form of syndromic diabetes caused by pathogenic variants in the WFS1 gene, which also leads to ER dysfunction.

1444-P: Knockdown of Etv5 Impairs Viability of Pancreatic Beta Cells by Enhancing ROS Production

Introduction: The transcription factor ETV5 regulates various biological processes such as cell proliferation and apoptosis, and activates a whole string of obesity- and diabetes-related genes. ETV5 is involved in tissue protection since mice with pancreatic loss of Etv5 show increased severity of pancreatitis and delayed tissue recovery. Here, we investigate the role of Etv5 knockdown (KD) on survival of injured endocrine pancreatic beta cells. Methods: We generated INS-1E beta cells with doxycycline (dox) -inducible lentiviral Etv5 sh-RNA knock-down (KD) . Cellular stress was induced by streptozotocin (STZ) and palmitate (PA) . We assessed viability (MTS) , apoptosis (caspases-3/7 activation) , necrosis (assay) , ER stress (BiP and CHOP gene expression) , oxidative stress (ROS production) and gene expression of SOD1, SOD2 and Catalase. Results: Etv5 KD significantly increases STZ/PA-induced loss of viability without changes in apoptosis but with increased necrosis. Etv5 KD did not change STZ/PA-induced gene expression of ER stress markers BiP and CHOP but significantly increased H2O2 production. This was associated with significantly elevated gene expression of mitochondrial SOD2, which converts superoxide into H2O2. Cytoplasmatic SOD1 and H2O2-decomposing catalase, PRDX1 and GPx1 remained unaffected. Conclusion: Increased circulating free fatty acids in obesity promote oxidative stress. Loss of ETV5 increases ROS production in mitochondria during cellular stress by PA and STZ, as indicated by upregulated SOD2 gene expression. This suggests a role for ETV5 in controlling mitochondrial oxidative stress in pancreatic β-cells. Disclosure X. Yi: None. G. Päth: None. J. Seufert: Advisory Panel; Abbott, Sanofi-Aventis Deutschland GmbH. Research Support; Boehringer Ingelheim International GmbH. Speaker's Bureau; Abbott Diabetes, AstraZeneca, Bayer AG, Boehringer Ingelheim International GmbH, Lilly, Novo Nordisk, Sanofi-Aventis Deutschland GmbH. Funding XY is supported by China Scolarship Council (CSC) China,GP received Projektförderung from the German Diabetes Society (DDG)

Protection of pancreatic beta cells against high glucose-induced toxicity by astaxanthin-s-allyl cysteine diester: alteration of oxidative stress and apoptotic-related protein expression

Purpose: High glucose (HG)-induced oxidative stress is associated with apoptosis in pancreatic β-cells. The protective effect of astaxanthin-s-allyl cysteine diester (AST-SAC) against HG-induced oxidative stress in pancreatic β-cells (βTC-tet cell line) in in vitro was studied. Materials and Methods: βTC-tet cell line was exposed to HG in the presence and absence of AST-SAC. Various parameters such as cell viability, reactive oxygen species generation, mitochondrial membrane potential, DNA fragmentation and expression of proteins involved in apoptosis [p53, B-cell lymphoma 2 (Bcl-2), Bcl-2 associated X (Bax), cytochrome c and caspase 3] were studied. Results: Pre-treatment of βTC-tet cells with AST-SAC (4, 8 and 12 μg/ml) in the presence of HG (25 mM) protected the viability of the cells in a dose-dependent manner. AST-SAC treatment mitigated the oxidative stress thereby preventing the mitochondrial dysfunction, DNA damage and apoptosis in βTC-tet cells against HG toxicity. Treatment with AST-SAC prevented the increased expression of p53 under HG conditions. Further, AST-SAC treatment maintained the level of pro-apoptotic (Bax, cleaved caspase-3 and cytochrome c) and anti-apoptotic (Bcl-2) proteins to that of the control level under HG exposed conditions in βTC-tet cells. Conclusion: Altogether, AST-SAC alleviated HG-induced oxidative damage and apoptosis in pancreatic β-cells by enhancing the antioxidant status and altering apoptotic-related protein expression.

Lipotoxicity and β-Cell Failure in Type 2 Diabetes: Oxidative Stress Linked to NADPH Oxidase and ER Stress.

A high caloric intake, rich in saturated fats, greatly contributes to the development of obesity, which is the leading risk factor for type 2 diabetes (T2D). A persistent caloric surplus increases plasma levels of fatty acids (FAs), especially saturated ones, which were shown to negatively impact pancreatic β-cell function and survival in a process called lipotoxicity. Lipotoxicity in β-cells activates different stress pathways, culminating in β-cells dysfunction and death. Among all stresses, endoplasmic reticulum (ER) stress and oxidative stress have been shown to be strongly correlated. One main source of oxidative stress in pancreatic β-cells appears to be the reactive oxygen species producer NADPH oxidase (NOX) enzyme, which has a role in the glucose-stimulated insulin secretion and in the β-cell demise during both T1 and T2D. In this review, we focus on the acute and chronic effects of FAs and the lipotoxicity-induced β-cell failure during T2D development, with special emphasis on the oxidative stress induced by NOX, the ER stress, and the crosstalk between NOX and ER stress.

Dietary (-)-epicatechin mitigates high fat-induced apoptosis, oxidative and endoplasmic reticulum stress in pancreatic β-cells both in vivo and in vitro

Dietary (-)-epicatechin mitigates high fat-induced apoptosis, oxidative and endoplasmic reticulum stress in pancreatic β-cells both in vivo and in vitro

Effect of Caesalpinia bonduc Polyphenol Extract on Alloxan-Induced Diabetic Rats in Attenuating Hyperglycemia by Upregulating Insulin Secretion and Inhibiting JNK Signaling Pathway.

Caesalpinia bonduc has been used in herbal medicines for the treatment of a wide range of diseases from decades. The present study has explored the remedial potential and underlying mechanism of polyphenol extract of Caesalpinia bonduc in alloxanized diabetic rats. HPLC/MS analysis confirmed the presence of phenolics in considerable concentrations in Caesalpinia bonduc extract. Administration of different doses (250 and 500 mg/kg) of CPP extract to hyperglycemic rats for 8 weeks restored blood and serum glucose, insulin, glycosylated hemoglobin, leptin, amylin, and carbohydrate metabolizing enzymes level towards normal compared to alloxanized diabetic group. The effect of CPP extract on various genes such as Pdx-1, Ins-1, ngn-3, GLUT-4, and IRS-1 in insulin signaling pathway and Traf-4, Traf-6, and Mapk-8 in MAPK downstream JNK cascade was examined through qRT-PCR to access the core molecular mechanism involved in CPP-induced recovery of diabetes. Results have revealed that CPP extract reduced oxidative stress in pancreatic β cells by restoring free radical scavenging potential, reducing the mRNA expression of Mapk-8, Traf-4, and Traf-6, and increasing the Pdx-1, Ins-1, ngn-3, GLUT-4, and IRS-1 expression ensuing regeneration of β cells and subsequent insulin release from pancreas. The results obtained in this study recommend that CPP extract may be a promising therapeutic restorative agent in the treatment of diabetes mellitus.

Nitric Oxide and Redox State Measurements in Pancreatic Beta Cells.

The role of oxidative stress in the pathogenesis of type 2 diabetes (T2D), especially pancreatic β-cell dysfunction and death, has become apparent in the last two decades. Peroxidase- and catalase-based antioxidant mechanisms are particularly weak in β-cells and can be easily overwhelmed by excessive production of reactive oxygen and nitrogen species in the course of pathological processes. Recent research has attempted to define in detail the mechanistic aspects of oxidative stress-induced β-cell dysfunction. Here, we describe the procedures for the measurement of various parameters important to assess oxidative stress in pancreatic β-cells. Detailed protocols for determination of nitric oxide (NO) production, the glutathione redox status, and general oxidative status in β-cells are presented in this chapter.

Islet Beta-Cell Specific Deletion of UBL5 Gene Associated with Mitochondrial Stress Leads to Diabetes in Mice and Beta-Cell Impairment/Death

Hyperglycaemia has been shown to cause oxidative stress in pancreatic β-cells, leading to activation of stress responses such as the mitochondrial unfolded protein response (UPRmt). Failure of these responses to adapt to or repair damage from stress results in dysfunction and death of β-cells. Ubiquitin-like protein 5 (UBL-5) is a protein known to have a crucial role in the UPRmt in C. elegans and upregulated during the UPRmt mammals. The aim of this study was to determine whether UBL-5 has a role in maintaining β-cell mass and function through modulating the UPRmt, by generating and characterising tamoxifen-inducible islet β-cell specific UBL-5 knockout mice. Homozygous β-cell UBL-5 knockout mice (UBL5-/-) showed glucose intolerance and lower plasma insulin levels during the OGTT while heterozygous mice (UBL5+/-) showed no difference. UBL5-/- had significantly reduced plasma insulin levels during IVGTT compared to control. Interestingly, UBL5+/- had significantly increased plasma insulin levels during IVGTT. Beta-cell mass was also significantly reduced in UBL5-/-, with most showing signs of frank diabetes (blood glucose >20 mM), polyuria and polydipsia, while UBL5+/- had increased β-cell mass. One week post UBL5 deletion, UBL5-/- mice had significantly increased blood glucose levels and islet cleaved caspase-3 levels compared to controls despite no difference in β-cell mass. In addition islets taken from UBL5-/- mice 1 week post UBL5 gene deletion showed significant decrease in insulin secretion, suggesting that β-cell dysfunction precedes the decrease in β-cell mass. Islets taken from UBL5+/- mice had increased insulin secretion compared to control. Real time PCR data showed a decrease in UPRmt genes (Clpx, Clpp, CHOP, Lonp1, HSP10, HSP70, ATF5) with HSP60 showing increased expression in the UBL5-/- mice one week after gene deletion, while the UBL5+/- mice showed an overall increase in most UPRmt related genes. Disclosure C. Haralambous: None. V. Ntouma: None.

ER stress attenuation by Aloe-derived polysaccharides in the protection of pancreatic β-cells from free fatty acid-induced lipotoxicity

Insulin resistance, a pathophysiology of type 2 diabetes, is associated with obesity. Lipotoxicity in obesity leads to the dysfunction and death of pancreatic β-cells and inadequate insulin production, thereby aggravating type 2 diabetes. The present study was conducted to determine the effect of Aloe vera polysaccharides (APs) as an anti-hyperglycemic agent and their mechanisms of action. Gel polysaccharides from Aloe extracts were separated using ultrafiltration devices with molecular weight-cutoff membranes, and the protective effect of APs on pancreatic β-cells in response to free fatty acids (FFAs) was determined. Hamster pancreatic β-cell line HIT-T15 was treated with palmitate and APs to analyze cellular responses. We observed a large number of apoptotic β-cell death after treatment with high levels of palmitate, but this was efficiently prevented by the addition of APs in a dose-dependent manner. It was found that the anti-apoptotic properties of APs were largely due to the relief of endoplasmic reticulum (ER) stress signaling. APs were effective in interfering with the FFA-induced activation of the PERK and IRE1 pathways as well as ROS generation, thereby protecting pancreatic β-cells from lipotoxicity. Although variation in the chain length of APs can influence the activity of FFA-mediated ER stress signaling in different ways, polysaccharide mixtures with molecular weights higher than 50 kDa showed greater antiapoptotic and antioxidant activity in β-cells. After oral administration of APs, markedly lowering fasting blood glucose levels were observed in db/db mice, providing evidence of the potential of APs as an alternative insulin sensitizer. Therefore, it was concluded that APs have a protective effect against type 2 diabetes by modulating obesity-induced ER stress in pancreatic β-cells.

Apoptosis signal regulating kinase-1 and NADPH oxidase mediate human amylin evoked redox stress and apoptosis in pancreatic beta-cells

Misfolded toxic human islet amyloid polypeptide or amylin (hA) and plasma membrane-associated redox complex, NADPH oxidase (NOX), have been implicated in the islet β-cell demise associated with type-2 diabetes mellitus (T2DM). Studies show that hA accumulation is stressful to β-cells and that misfolding of human amylin evokes redox stress and activates mitogen activated protein (MAP) kinases, p38 MAPK and c-Jun N-terminal (JNK) kinase. However, the molecular link and causality between hA-evoked redox stress, NOX activity and MAP kinases signaling in pancreatic β-cells is incompletely understood. Here, we show that in the process of activating JNK, aggregation prone hA also activates an upstream apoptosis signal regulating kinase-1 (ASK1) with concomitant decrease in intracellular levels of reduced glutathione. Inhibition of ASK1 kinase activity, either by specific ASK1 inhibitor, NQDI1 or by thiol antioxidants reduces human amylin-evoked ASK1 and JNK activation and consequently human amylin toxicity in rat insulinoma Rin-m5F cells and human islets. β-cell specific overexpression of human amylin in mouse islets elicited ASK1 phosphorylation and activation in β-cells but not in other rodent's islet or exocrine cells. This ASK1 activation strongly correlated with islet amyloidosis and diabetes progression. Cytotoxic human amylin additionally stimulated pro-oxidative activity and expressions of plasma membrane bound NADPH oxidase (NOX) and its regulatory subunits. siRNA mediated NOX1 knockdown and selective NOX inhibitors, ML171 and apocynin, significantly reduced hA-induced mitochondrial stress in insulinoma beta-cells. However, NOX inhibitors were largely ineffective against hA-evoked redox stress and activation of cytotoxic ASK1/JNK signaling complex. Thus, our studies suggest that NOX1 and ASK1 autonomously mediate human amylin-evoked redox and mitochondrial stress in pancreatic β-cells.

Pioglitazone Attenuates Palmitate-Induced Inflammation and Endoplasmic Reticulum Stress in Pancreatic β-Cells.

BackgroundThe nuclear receptor peroxisome proliferator-activator gamma (PPARγ) is a useful therapeutic target for obesity and diabetes, but its role in protecting β-cell function and viability is unclear.MethodsTo identify the potential functions of PPARγ in β-cells, we treated mouse insulinoma 6 (MIN6) cells with the PPARγ agonist pioglitazone in conditions of lipotoxicity, endoplasmic reticulum (ER) stress, and inflammation.ResultsPalmitate-treated cells incubated with pioglitazone exhibited significant improvements in glucose-stimulated insulin secretion and the repression of apoptosis, as shown by decreased caspase-3 cleavage and poly (adenosine diphosphate [ADP]-ribose) polymerase activity. Pioglitazone also reversed the palmitate-induced expression of inflammatory cytokines (tumor necrosis factor α, interleukin 6 [IL-6], and IL-1β) and ER stress markers (phosphor-eukaryotic translation initiation factor 2α, glucose-regulated protein 78 [GRP78], cleaved-activating transcription factor 6 [ATF6], and C/EBP homologous protein [CHOP]), and pioglitazone significantly attenuated inflammation and ER stress in lipopolysaccharide- or tunicamycin-treated MIN6 cells. The protective effect of pioglitazone was also tested in pancreatic islets from high-fat-fed KK-Ay mice administered 0.02% (wt/wt) pioglitazone or vehicle for 6 weeks. Pioglitazone remarkably reduced the expression of ATF6α, GRP78, and monocyte chemoattractant protein-1, prevented α-cell infiltration into the pancreatic islets, and upregulated glucose transporter 2 (Glut2) expression in β-cells. Moreover, the preservation of β-cells by pioglitazone was accompanied by a significant reduction of blood glucose levels.ConclusionAltogether, these results support the proposal that PPARγ agonists not only suppress insulin resistance, but also prevent β-cell impairment via protection against ER stress and inflammation. The activation of PPARγ might be a new therapeutic approach for improving β-cell survival and insulin secretion in patients with diabetes mellitus

Editorial note: Integrating insulin secretion and ER stress in pancreatic β-cells

Editorial note: Integrating insulin secretion and ER stress in pancreatic β-cells

IRE1 inhibition perturbs the unfolded protein response in a pancreatic β-cell line expressing mutant proinsulin, but does not sensitize the cells to apoptosis.

BackgroundThe Akita mutation (C96Y) in the insulin gene results in early onset diabetes in both humans and mice. Expression of mutant proinsulin (C96Y) causes endoplasmic reticulum (ER) stress in pancreatic β-cells and consequently the cell activates the unfolded protein response (UPR). Since the proinsulin is terminally misfolded ER stress is irremediable and chronic activation of the UPR eventually activates apoptosis in some cells. Here we analyzed the IRE1-dependent activation of genes in response to misfolded proinsulin production in an inducible mutant proinsulin (C96Y) insulinoma cell line.ResultsThe IRE1 endoribonuclease inhibitors 4μ8c and MKC-3946 prevented the splicing of the XBP1 mRNA in response to ER stress caused by mutant proinsulin production. Microarray expression analysis and qPCR validation of select genes revealed that maximal upregulation of many UPR genes in response to mutant proinsulin production required IRE1, although most were still increased above control. Interestingly, neither degradation of misfolded proinsulin via ER-associated degradation (ERAD), nor apoptosis induced by prolonged misfolded proinsulin expression were affected by inhibiting IRE1.ConclusionsAlthough maximal induction of most UPR genes requires IRE1, inhibition of IRE1 does not affect ERAD of misfolded proinsulin or predispose pancreatic β-cells expressing misfolded proinsulin to chronic ER stress-induced apoptosis.

Effects of mulberry ethanol extracts on hydrogen peroxide-induced oxidative stress in pancreatic β-cells

Reactive oxygen species (ROS) are key mediators of mammalian cellular damage and are associated with diseases such as aging, arteriosclerosis, inflammation, rheumatoid arthritis and diabetes. Type 1 diabetes develops upon the destruction of pancreatic β-cells, which is partly due to ROS activity. In this study, we investigated the cytoprotective and anti-oxidative effects of fractionated mulberry extracts in mouse insulin-producing pancreatic β-cells (MIN6N cells). Treatment with hydrogen peroxide (H2O2) induced significant cell death and increased intracellular ROS levels, lipid peroxidation and DNA fragmentation in the MIN6N cells. Fractionated mulberry extracts significantly reduced the H2O2-dependent production of intracellular ROS, 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals and lipid peroxidation. In addition, mulberry extracts inhibited DNA fragmentation induced by H2O2. Thus, the antioxidant properties of mulberry extracts in pancreatic β-cells may be exploited for the prevention or treatment of type 1 diabetes.

Endoplasmic Reticulum Redox State Is Not Perturbed by Pharmacological or Pathological Endoplasmic Reticulum Stress in Live Pancreatic β-Cells

Accumulation of unfolded, misfolded and aggregated proteins in the endoplasmic reticulum (ER) causes ER stress. ER stress can result from physiological situations such as acute increases in secretory protein biosynthesis or pathological conditions that perturb ER homeostasis such as alterations in the ER redox state. Here we monitored ER redox together with transcriptional output of the Unfolded Protein Response (UPR) in INS-1 insulinoma cells stably expressing eroGFP (ER-redox-sensor) and mCherry protein driven by a GRP78 promoter (UPR-sensor). Live cell imaging, flow cytometry and biochemical characterization were used to examine these parameters in response to various conditions known to induce ER stress. As expected, treatment of the cells with the reducing agent dithiothreitol caused a decrease in the oxidation state of the ER accompanied by an increase in XBP-1 splicing. Unexpectedly however, other treatments including tunicamycin, thapsigargin, DL-homocysteine, elevated free fatty acids or high glucose had essentially no influence on the ER redox state, despite inducing ER stress. Comparable results were obtained with dispersed rat islet cells expressing eroGFP. Thus, unlike in yeast cells, ER stress in pancreatic β-cells is not associated with a more reducing ER environment.

Integrating insulin secretion and ER stress in pancreatic β-cells

After food consumption, insulin-secreting pancreatic β-cells detect increased glucose and incretin hormones, and respond by releasing insulin. Wolfram syndrome 1, a protein that mitigates endoplasmic reticulum (ER) stress, is now shown to regulate insulin synthesis and release--revealing a molecular point of convergence between the ER stress and insulin release pathways.

Susceptibility of glucokinase-MODY mutants to inactivation by oxidative stress in pancreatic β-cells.

OBJECTIVEThe posttranslational regulation of glucokinase (GK) differs in hepatocytes and pancreatic β-cells. We tested the hypothesis that GK mutants that cause maturity-onset diabetes of the young (GK-MODY) show compromised activity and posttranslational regulation in β-cells.RESEARCH DESIGN AND METHODSActivity and protein expression of GK-MODY and persistent hyperinsulinemic hypoglycemia of infancy (PHHI) mutants were studied in β-cell (MIN6) and non–β-cell (H4IIE) models. Binding of GK to phosphofructo-2-kinase, fructose-2,6-bisphosphatase (PFK2/FBPase2) was studied by bimolecular fluorescence complementation in cell-based models.RESULTSNine of 11 GK-MODY mutants that have minimal effect on enzyme kinetics in vitro showed decreased specific activity relative to wild type when expressed in β-cells. A subset of these were stable in non–β-cells but showed increased inactivation in conditions of oxidative stress and partial reversal of inactivation by dithiothreitol. Unlike the GK-MODY mutants, four of five GK-PHHI mutants had similar specific activity to wild type and Y214C had higher activity than wild type. The GK-binding protein PFK2/FBPase2 protected wild-type GK from oxidative inactivation and the decreased stability of GK-MODY mutants correlated with decreased interaction with PFK2/FBPase2.CONCLUSIONSSeveral GK-MODY mutants show posttranslational defects in β-cells characterized by increased susceptibility to oxidative stress and/or protein instability. Regulation of GK activity through modulation of thiol status may be a physiological regulatory mechanism for the control of GK activity in β-cells.