Rat Insulinoma Cells Research Articles

NLRP3 inflammasome mediates angiotensin II-induced islet β cell apoptosis.

Elevation of angiotensin II (Ang II) in the serum of patients with diabetes is known to promote apoptosis of islet β cells, but the underlying mechanism remains unclear. The aim of the present study was to explore the role of Nod-like receptor protein 3 (NLRP3) inflammasome in Ang II-induced apoptosis of pancreatic islet β cells and investigate the possible underlying mechanism. The effect of Ang II on INS-1 cell (a rat insulinoma cell line) viability was detected by CCK-8 method. The cell apoptosis was detected by flow cytometry and western blot analysis. The effect of Ang II on the expressions of thioredoxin-interacting protein (TXNIP) and NLRP3 protein was detected by western blot analysis. The expression of TXNIP mRNA was detected by real-time polymerase chain reaction. The results showed that Ang II was able to reduce INS-1 cell viability and promote apoptosis and at the same time up-regulate the expressions of TXNIP and NLRP3 components. Ang II-induced apoptosis was inhibited after administration of the NLRP3 inhibitor MCC950, and TXNIP silencing could reduce the NLRP3 expression and apoptosis, while both effects of Ang II on TXNIP-NLRP3 and its apoptosis-inducing effect were inhibited by angiotensin II type I receptor (AT1R) blocker Telmisartan. Our results demonstrated that the TXNIP-NLRP3 inflammasome pathway mediated Ang II-induced INS-1 cell apoptosis and might hopefully become a novel target for the treatment of diabetes mellitus.

Oleic acid protects insulin-secreting INS-1E cells against palmitic acid-induced lipotoxicity along with an amelioration of ER stress.

It is demonstrated that unsaturated fatty acids can counteract saturated fatty acids-induced lipotoxicity, but the molecular mechanisms are unclear. In this study, we investigated the protective effects of monounsaturated oleic acid (OA) against saturated palmitic acid (PA)-induced cytotoxicity in rat β cells as well as islets, and mechanistically focused on its regulation on endoplasmic reticulum (ER) stress. Rat insulinoma cell line INS-1E cells and primary islets were treated with PA with or without OA for 24 h to determine the cell viability, apoptosis, and ER stress. SD rats were fed with high-fat diet (HFD) for 16 w, then, HFD was half replaced by olive oil to observe the protective effects of monounsaturated fatty acids rich diet. We demonstrated that PA impaired cell viability and insulin secretion of INS-1E cells and rat islets, but OA robustly rescued cells from cell death. OA substantially alleviated either PA or chemical ER stressors (thapsigargin or tunicamycin)-induced ER stress. Importantly, OA attenuated the activity of PERK-eIF2α-ATF4-CHOP pathway and regulated the ER Ca2+ homeostasis. In vivo, only olive oil supplementation did not cause significant changes, while high-fat diet (HFD) for 32 w obviously induced islets ER stress and impaired insulin sensitivity in SD rats. Half replacement of HFD with olive oil (a mixed diet) has ameliorated this effect. OA alleviated PA-induced lipotoxicity in INS-1E cells and improved insulin sensitivity in HFD rats. The amelioration of PA triggered ER stress may be responsible for its beneficial effects in β cells.

The effect of anodal/cathodal biphasic electrical stimulation on insulin release.

We studied the effects of electrical stimulation on insulin release from rat insulinoma (INS-1) cells. The anodal/cathodal biphasic stimulation (ACBPS) electrical waveform resulted in a voltage- and stimulation duration-dependent increase in insulin release. ACBPS elicited insulin release both in the presence and absence of glucose. Basal and ACBPS-induced insulin secretion could be inhibited by mitochondrial poisons and calcium channel blockers, indicating that insulin release was dependent on adenosine triphosphate (ATP) and the influx of calcium. ACBPS parameters that released insulin caused no detectable plasma membrane damage or cytotoxicity, although temporary morphological changes could be observed immediately after ACBPS. ACBPS did not alter the plasma membrane transmembrane potential but did cause pronounced uptake of MitoTracker Red into the mitochondrial membrane, indicating an increased mitochondrial membrane potential. While the ATP:ADP ratio after ACBPS did not change, the guanosine triphosphate (GTP) levels increased and increased GTP levels have previously been associated with insulin release in INS-1 cells. These results provide evidence that ACBPS can have significant biological effects on cells. In the case of INS-1 cells, ACBPS promotes insulin release without causing cytotoxicity.

Phosphorylation-Dependent Intra-Domain Interaction of the Cx37 Carboxyl-Terminus Controls Cell Survival.

Differential phosphorylation of the carboxyl-terminus of connexin 37 (Cx37-CT) regulates phenotypic switching between cell growth phenotypes (cell death, cell cycle arrest, proliferation). The specific phosphorylation events in the Cx37-CT that are necessary for these growth regulatory effects are currently unknown. Through the combined use of deletion and site specific (de)phospho-mimetic Cx37-CT mutants, our data suggest a phosphorylation-dependent interaction between the mid-tail (aa 273–317) and end-tail (aa 318–333) portions of the Cx37-CT that regulates cell survival. As detected by mass spectrometry, Cx37 was phosphorylated at serines 275, 321, and 328; phosphomimetic mutations of these sites resulted in cell death when expressed in rat insulinoma cells. Alanine substitution at S328, but not at S275 or S321, also triggered cell death. Cx37-S275D uniquely induced the death of only low density, non-contact forming cells, but neither hemichannel open probability nor channel conductance distinguished death-inducing mutants. As channel function is necessary for cell death, together the data suggest that the phosphorylation state of the Cx37-CT controls an intra-domain interaction within the CT that modifies channel function and induces cell death.

Endoplasmic Reticulum-Associated Degradation (ERAD) Has a Critical Role in Supporting Glucose-Stimulated Insulin Secretion in Pancreatic β-Cells.

The molecular underpinnings of β-cell dysfunction and death leading to diabetes are not fully elucidated. The objective of the current study was to investigate the role of endoplasmic reticulum-associated degradation (ERAD) in pancreatic β-cells. Chemically induced ERAD deficiency in the rat insulinoma cell line INS-1 markedly reduced glucose-stimulated insulin secretion (GSIS). The mechanistic basis for this effect was studied in cells and mice lacking ERAD as a consequence of genetic ablation of the core ERAD protein SEL1L. Targeted disruption of SEL1L in INS-1 cells and in mouse pancreatic β-cells impaired ERAD and led to blunted GSIS. Additionally, mice with SEL1L deletion in β-cells were chronically hyperglycemic after birth and increasingly glucose intolerant over time. SEL1L absence caused an entrapment of proinsulin in the endoplasmic reticulum compartment in both INS-1 cells and mouse pancreatic β-cells. Both folding-competent and folding-deficient proinsulin can physiologically interact with and be efficiently degraded by HRD1, the E3 ubiquitin ligase subunit of the ERAD complex. GSIS impairment in insulinoma cells was accompanied by a reduced intracellular Ca2+ ion level, overproduction of reactive oxygen species, and lowered mitochondrial membrane potential. Together, these findings suggest that ERAD plays a pivotal role in supporting pancreatic β-cell function by targeting wild-type and folding-deficient proinsulin for proteosomal degradation. ERAD deficiency may contribute to the development of diabetes by affecting proinsulin processing in the ER, intracellular Ca2+ concentration, and mitochondrial function.

Antidiabetic Activity of Phytosaponin in STZ-Induced Type I Diabetes in Rats

Objective: The study was focused on antidiabetic activity of saponin isolated from the fruits of Momordica dioica against STZ induced Type I diabetic mellitus in rats. Methods: Saponin was isolated from methanolic extract of fruits of M. dioica and purification was achieved through fractionation method of TLC. Overnight fasting, for Type I diabetes was induced in rats by I.P. of STZ dose of 65 mg/kg b.w. During the study, body weight and fasting blood glucose level were taken. At the end of study, animals in all groups were sacrificed and biochemical parameters; Lipid profile, HbA1c, Serum insulin, pancreatic insulin, histology of pancreas, and in-vitro insulin secretion activity and proliferation of beta cells in RIN-5F cell lines were performed. Results and conclusion: Significant antidiabetic activity of saponin Momordica dioica (SMD) observed in the present investigation could be the result of decreased blood glucose levels, improved body mass, showed improvisation in lipid profile. The diabetic rats were validated by their higher HbA1c levels as compared the normal control but the treated diabetic group showed significantly decreased in the HbA1c levels. Similarly the increase in serum insulin and pancreatic insulin may facilitate to prevent diabetic complication. Diabetic pancreas of rats showed degeneration of beta cell density and disruption of normal architecture, whereas treatment group showed reversible beta cell degeneration. SMD was found to possess insulinotropic activity when rat insulinoma cell line was treated with it. Proliferation of RIN-5F was found to increase at low doses but tend to decrease with accelerating doses.

Mitochondrial lysine deacetylation promotes energy metabolism and calcium signaling in insulin-secreting cells.

In pancreatic β-cells, mitochondria generate signals that promote insulin granule exocytosis. Here we study how lysine acetylation of mitochondrial proteins mechanistically affects metabolism-secretion coupling in insulin-secreting cells. Using mass spectrometry-based proteomics, we identified lysine acetylation sites in rat insulinoma cell line clone 1E cells. In cells lacking the mitochondrial lysine deacetylase sirtuin-3 (SIRT3), several matrix proteins are hyperacetylated. Disruption of the SIRT3 gene has a deleterious effect on mitochondrial energy metabolism and Ca2+ signaling. Under resting conditions, SIRT3 deficient cells are overactivated, which elevates the respiratory rate and enhances calcium signaling and basal insulin secretion. In response to glucose, the SIRT3 knockout cells are unable to mount a sustained cytosolic ATP response. Calcium signaling is strongly reduced and the respiratory response as well as insulin secretion are blunted. We propose mitochondrial protein lysine acetylation as a control mechanism in β-cell energy metabolism and Ca2+ signaling.-De Marchi, U., Galindo, A. N., Thevenet, J., Hermant, A., Bermont, F., Lassueur, S., Domingo, J. S., Kussmann, M., Dayon, L., Wiederkehr, A. Mitochondrial lysine deacetylation promotes energy metabolism and calcium signaling in insulin-secreting cells.

Prolonged Exposure to Insulin Inactivates Akt and Erk1/2 and Increases Pancreatic Islet and INS1E β-Cell Apoptosis.

Chronic hyperinsulinemia, in vivo, increases the resistance of peripheral tissues to insulin by desensitizing insulin signaling. Insulin, in a heterologous manner, can also cause IGF-1 resistance. The aim of the current study was to investigate whether insulin-mediated insulin and IGF-1 resistance develops in pancreatic β-cells and whether this resistance results in β-cell decompensation. Chronic exposure of rat islets or INS1E β-cells to increasing concentrations of insulin decreased AktS473 phosphorylation in response to subsequent acute stimulation with 10 nM insulin or IGF-1. Prolonged exposure to high insulin levels not only inhibited AktS473 phosphorylation, but it also resulted in a significant inhibition of the phosphorylation of P70S6 kinase and Erk1/2 phosphorylation in response to the acute stimulation by glucose, insulin, or IGF-1. Decreased activation of Akt, P70S6K, and Erk1/2 was associated with decreased insulin receptor substrate 2 tyrosine phosphorylation and insulin receptor β-subunit abundance; neither IGF receptor β-subunit content nor its phosphorylation were affected. These signaling impairments were associated with decreased SERCA2 expression, perturbed plasma membrane calcium current and intracellular calcium handling, increased endoplasmic reticulum stress markers such as eIF2αS51 phosphorylation and Bip (GRP78) expression, and increased islet and β-cell apoptosis. We demonstrate that prolonged exposure to high insulin levels induces not only insulin resistance, but in a heterologous manner causes resistance to IGF-1 in rat islets and insulinoma cells resulting in decreased cell survival. These findings suggest the possibility that chronic exposure to hyperinsulinemia may negatively affect β-cell mass by increasing β-cell apoptosis.

PEP-1-paraoxonase 1 fusion protein prevents cytokine-induced cell destruction and impaired insulin secretion in rat insulinoma cells

Pancreatic beta cell destruction and dysfunction induced by cytokines is a major cause of type 1 diabetes. Paraoxonase 1 (PON1), an arylesterase with antioxidant activity, has been shown to play an important role in preventing the development of diabetes in transgenic mice. However, no studies have examined the anti-diabetic effect of PON1 delivered to beta cells using protein transduction. In this study, we expressed the cell-permeable PON1 fused with PEP-1 protein transduction domain (PEP-1-PON1) to investigate whether transduced PEP-1-PON1 protects beta cells against cytokine-induced cytotoxicity. PEP-1-PON1 was effectively delivered to INS-1 cells and prevented cytokine-induced cell destruction in a dose-dependent manner. Transduced PEP-1-PON1 significantly reduced the levels of reactive oxygen species (ROS) and nitric oxide (NO), DNA fragmentation, and expression of inflammatory mediators, endoplasmic reticulum (ER) stress proteins, and apoptosis-related proteins in cytokine-treated cells. Moreover, transduced PEP-1-PON1 restored the decrease in basal and glucose-stimulated insulin secretion induced by cytokines. These data indicate that PEP-1-PON1 protects beta cells from cytokine-induced cytotoxicity by alleviating oxidative/nitrosative stress, ER stress, and inflammation. Thus, PEP-1-mediated PON1 transduction might be an effective method to reduce the extent of destruction and dysfunction of pancreatic beta cells in autoimmune diabetes.

Hydroxysafflor yellow A attenuates high glucose-induced pancreatic β-cells oxidative damage via inhibiting JNK/c-jun signaling pathway

Pancreatic β-cells apoptosis and dysfunction induced by glucose toxicity were attributed to the formation of excess oxidative damage. Some studies have found that hydroxysafflor yellow A has strong effects to scavenge oxidative stress and inhibit apoptosis. In order to explore the influence of HSYA on oxidative stress induced by high glucose and the potential mechanisms, we set up a high glucose damage model and induced oxidative stress in INS-1 rat insulinoma cells. N-acetylcysteine was added as a group of oxidative stress scavenger. After 72 h of cultivation, the related indexes of oxidative stress (reactive oxygen species, catalase, glutathione peroxidase, lipid peroxidation, and superoxide dismutase), apoptosis (caspase3, parp) and the function of glucose stimulated insulin secretion were determined. In addition, the signaling pathway proteins of C-Jun NH2 -terminal kinases (JNK), phosphorylated JNK, C-jun, phosphorylated C-jun were evaluated. Fluorescence microscopy, qRT-PCR, western blotting were the main methods used in the experiment. Our results showed that hydroxysafflor yellow A reduced pancreatic β-cells apoptosis by attenuating oxidative damage, and JNK/c-Jun signaling pathway was involved. It indicated a significant mechanism for the positive impacts of HSYA on oxidative stress induced by high glucose, and provide important basis for using HSYA in diabetic prevention and therapy.

Impaired Store-Operated Calcium Entry and STIM1 Loss Lead to Reduced Insulin Secretion and Increased Endoplasmic Reticulum Stress in the Diabetic β-Cell.

Store-operated Ca2+ entry (SOCE) is a dynamic process that leads to refilling of endoplasmic reticulum (ER) Ca2+ stores through reversible gating of plasma membrane Ca2+ channels by the ER Ca2+ sensor Stromal Interaction Molecule 1 (STIM1). Pathogenic reductions in β-cell ER Ca2+ have been observed in diabetes. However, a role for impaired SOCE in this phenotype has not been tested. We measured the expression of SOCE molecular components in human and rodent models of diabetes and found a specific reduction in STIM1 mRNA and protein levels in human islets from donors with type 2 diabetes (T2D), islets from hyperglycemic streptozotocin-treated mice, and INS-1 cells (rat insulinoma cells) treated with proinflammatory cytokines and palmitate. Pharmacologic SOCE inhibitors led to impaired islet Ca2+ oscillations and insulin secretion, and these effects were phenocopied by β-cell STIM1 deletion. STIM1 deletion also led to reduced ER Ca2+ storage and increased ER stress, whereas STIM1 gain of function rescued β-cell survival under proinflammatory conditions and improved insulin secretion in human islets from donors with T2D. Taken together, these data suggest that the loss of STIM1 and impaired SOCE contribute to ER Ca2+ dyshomeostasis under diabetic conditions, whereas efforts to restore SOCE-mediated Ca2+ transients may have the potential to improve β-cell health and function.

Magnetoliposomes as Contrast Agents for Longitudinal in vivo Assessment of Transplanted Pancreatic Islets in a Diabetic Rat Model

Magnetoliposomes (MLs) were synthesized and tested for longitudinal monitoring of transplanted pancreatic islets using magnetic resonance imaging (MRI) in rat models. The rat insulinoma cell line INS-1E and isolated pancreatic islets from outbred and inbred rats were used to optimize labeling conditions in vitro. Strong MRI contrast was generated by islets exposed to 50 µg Fe/ml for 24 hours without any increased cell death, loss of function or other signs of toxicity. In vivo experiments showed that pancreatic islets (50–1000 units) labeled with MLs were detectable for up to 6 weeks post-transplantation in the kidney subcapsular space. Islets were also monitored for two weeks following transplantation through the portal vein of the liver. Hereby, islets labeled with MLs and transplanted under the left kidney capsule were able to correct hyperglycemia and had stable MRI signals until nephrectomy. Interestingly, in vivo MRI of streptozotocin induced diabetic rats transplanted with allogeneic islets demonstrated loss of MRI contrast between 7–16 days, indicative of loss of islet structure. MLs used in this study were not only beneficial for monitoring the location of transplanted islets in vivo with high sensitivity but also reported on islet integrity and hereby indirectly on islet function and rejection.

A cell-based assay for the detection of pathogenic anti-voltage-gated calcium channel autoantibodies in immunoglobulin G from patients with type 1 diabetes

Recent studies have postulated the presence of functional autoantibodies (Abs) against L-type voltage gated calcium channels (VGCCs) in the serum of patients with type 1 diabetes, with various proposed physiological consequences, both islet cell associated and extra-glandular. Arguably, the most potentially damaging effect reported for these Abs is induction of apoptosis in pancreatic beta (β) cells, yet a convincing pathogenic mechanism remains to be demonstrated. In the current study, we report an assay of reactive oxygen species (ROS) stress induction in the rat insulinoma cell line Rin A12, as determined by 2′, 7′-Dichlorofluorescein diacetate (DCF-DA) fluorescence detection by flow cytometry. We demonstrate that incubation of Rin A12 cells with immunoglobulin G (IgG) containing anti-VGCC activity from patients with T1D mediates a significant increase in ROS, with subsequent induction of apoptosis, as determined by positivity for annexin V expression. Neither T1D patient-derived IgG lacking anti-VGCC activity or IgG from healthy donors altered ROS or annexin V expression, indicating the new assay is specific for the detection of functional anti-VGCC Abs. Subsequent screening of IgG samples derived from individual patients indicated a prevalence of approximately 75% in a cohort of 20 patients with T1D. The new cell-based assay provides, for the first time, experimental evidence supporting a plausible pathophysiological mechanism underlying anti-VGCC Ab-mediated apoptosis induction in β cells. Additionally, the assay is a considerable advance on previously published methods for detecting and characterising the functional activity of anti-VGCC Abs in patient-derived samples.

Functional Characterization of Wolfram Syndrome 1 Protein in ß-Cell Function and Viability

Endoplasmic reticulum (ER) homeostasis is crucial for proper β-cell function as evidenced by rare monogenic diabetic disorders, such as Wolfram syndrome, arising from genetic defects in key ER molecules. Wolfram syndrome stems from mutation of the ER transmembrane protein, Wolfram Syndrome 1 (WFS1), resulting in a recessive, progressive neurodegenerative disorder that first manifests as juvenile-onset diabetes mellitus (DM). While many WFS1 variants are associated with DM, the role of WFS1 in β-cell viability and function remains unclear. Our central hypothesis is that WFS1 regulates β-cell viability through downregulation of ER stress-responsive pro-apoptotic factors and β-cell function through modulation of key ER Ca2+ transporters. To test this hypothesis, we generated inducible β-cell models of WFS1 knockdown and overexpression using rat insulinoma cell lines to monitor β-cell function and β-cell death. We employed chemical and physiologic stimuli to model ER stress and monitored its downstream effects by real-time PCR, immunoblot and intracellular calcium mobilization. Our data indicate that β-cells depleted of WFS1 exhibit impaired insulin secretion and reduced insulin content. Additionally, knockdown of WFS1 is associated with decreased ER Ca2+, increased cytosolic Ca2+ and increased β-cell death. Conversely, increasing WFS1 expression in vitro increases insulin production and confers protection against ER stress-mediated β-cell death. Our data suggest that WFS1 may preserve β-cell viability by reducing the expression of pro-apoptotic factors CHOP and TRIB3. Future studies seek to clarify the mechanisms by which WFS1 protects β-cells against metabolic stressors and promotes insulin production. These studies will expand our understanding of the broader mechanisms by which ER dysfunction triggers β-cell pathology in more common forms of diabetes, and provide potential novel targets for intervention that center on preserving ER homeostasis. Disclosure D. Abreu: None. Z. Lavagnino: None. C.M. Brown: None. D.W. Piston: None. F. Urano: Research Support; Self; Eli Lilly and Company. Stock/Shareholder; Self; CytRx.

Targeting Cellular Calcium Homeostasis to Prevent Beta-Cell Death in Type 1 Diabetes

Cytokines are important mediators of beta cell death leading to type 1 diabetes (T1DM). The mechanism of cytokine-mediated beta cell death has been previously shown to involve alterations in beta cell calcium homeostasis and increases in endoplasmic reticulum (ER) stress. Our group has previously established that pharmacologic modulation of cellular calcium levels with dantrolene and sitagliptin can significantly decrease cytokine and ER stress-mediated activation of the calpain and thioredoxin interacting protein pro-apoptotic pathways and suppress cell death in vitro using INS-1E rat insulinoma cells. The current study was designed to determine if dantrolene and sitagliptin can prevent beta cell death and diabetes in the low dose streptozotocin (STZ) model of T1DM. Male C57Bl/6 mice were treated with 5 doses of STZ (50 mg/kg) daily by 10 days of intraperitoneal (IP) injections of placebo, dantrolene, or sitagliptin. Blood sugar was monitored via tail vein blood using a glucometer. Blood glucose and insulin levels in response to 2g/kg IP dextrose injection were obtained on day 10. Unmethylated insulin DNA was also measured from plasma drawn on day 0, 8 and 15 to monitor beta cell death. Sitagliptin treated mice were resistant to low dose STZ induced diabetes as indicated by lower random blood glucose levels and normalization of blood glucose and insulin levels in response to IP dextrose infusion compared to placebo treated animals. In addition, sitagliptin treated animals had significantly lower levels of circulating unmethylated insulin DNA suggesting lower incidence of beta cell death as compared to placebo treated animals. In summary, in the low dose STZ model of diabetes, sitagliptin lowered random blood sugar, normalized blood glucose and insulin response to IP glucose tolerance testing and significantly decreased STZ mediated beta cell death. This study supports further study on the effect of sitagliptin and DPP-4 inhibition on beta cell function and survival in T1DM. Disclosure A.L. Clark: None. D. Abreu: None. C.M. Brown: None. F. Urano: Research Support; Self; Eli Lilly and Company. Stock/Shareholder; Self; CytRx.

The Effect of nAChR Signaling on Terminal-Unfolded Protein Response under Endoplasmic Reticulum Stress in INS1 Cells

Background: Pancreatic β-cells are specialized for the production of insulin and susceptible to endoplasmic reticulum (ER) stress. Irremediable ER stress is coupled with terminal-unfolded protein response (T-UPR) through activating IRE1α, which leads to β-cell dysfunction and apoptosis. Previous reports show the effects of nAChR signaling against ER stress-induced neurotoxicity. However, it is still unknown if and how nAChR signaling affects T-UPR in β-cells. The aim of the study is to determine if and how nAChR signaling reduces T-UPR by regulating IRE1α activity in β-cells. Methods: We investigate the role of nAChR signaling on T-UPR signaling under ER stress or specific IRE1αactivation using rat insulinoma cell line, INS-1 cells. Cells were treated with Nicotine; an agonist of nAChR, α-Bungarotoxin(α-BTX); an antagonist of nAChR α7 subunit, and Tunicamycin(Tm) and Thapsigargin(Tg); ER stress inducers. Expression levels of spliced XBP1 and TXNIP/Insulin1 (Proinsulin), as T-UPR markers, were analyzed by real time PCR and/or Western blotting. Results: We first confirmed the expression levels of nAChR α7 subunits in INS1 cells. ER stress inducers (Tm or Tg) increased XBP1 splicing, and Nicotine (>10 μM) reduced it. Nicotine reversed ER stress-induced increase of TXNIP and decrease of Insulin1 (proinsulin) expression in both mRNA and protein levels. Further, Nicotine reversed increase of TXNIP and decrease of Insulin1 mRNA expression levels induced by IRE1α overexpression in Dox-inducible wild type IRE1α-overexpressing INS1 cells. Those effects of Nicotine were canceled by α-BTX. Conclusion: Our results suggest that nAChR signaling could prevent T-UPR induced by ER stress in INS1 cells. Disclosure T. Ishibashi: None. S. Morita: None. A. Doi: None. H. Iwakura: None. H. Ariyasu: None. M. Nishi: None. H. Furuta: None. T. Akamizu: None.

Site-specific glycations of apolipoprotein A-I lead to differentiated functional effects on lipid-binding and on glucose metabolism

Prolonged hyperglycemia in poorly controlled diabetes leads to an increase in reactive glucose metabolites that covalently modify proteins by non-enzymatic glycation reactions. Apolipoprotein A-I (apoA-I) of high-density lipoprotein (HDL) is one of the proteins that becomes glycated in hyperglycemia. The impact of glycation on apoA-I protein structure and function in lipid and glucose metabolism were investigated.ApoA-I was chemically glycated by two different glucose metabolites (methylglyoxal and glycolaldehyde). Synchrotron radiation and conventional circular dichroism spectroscopy were used to study apoA-I structure and stability. The ability to bind lipids was measured by lipid-clearance assay and native gel analysis, and cholesterol efflux was measured by using lipid-laden J774 macrophages. Diet induced obese mice with established insulin resistance, L6 rat and C2C12 mouse myocytes, as well as INS-1E rat insulinoma cells, were used to determine in vivo and in vitro glucose uptake and insulin secretion.Site-specific, covalent modifications of apoA-I (lysines or arginines) led to altered protein structure, reduced lipid binding capability and a reduced ability to catalyze cholesterol efflux from macrophages, partly in a modification-specific manner. The stimulatory effects of apoA-I on the in vivo glucose clearance were negatively affected when apoA-I was modified with methylglyoxal, but not with glycolaldehyde. The in vitro data showed that both glucose uptake in muscle cells and insulin secretion from beta cells were affected. Taken together, glycation modifications impair the apoA-I protein functionality in lipid and glucose metabolism, which is expected to have implications for diabetes patients with poorly controlled blood glucose.

The LIM homeodomain protein ISL1 mediates the function of TCF7L2 in pancreatic beta cells.

Pancreatic β-cell Tcf7l2 deletion or its functional knockdown suggested the essential role of this Wnt pathway effector in controlling insulin secretion, glucose homeostasis and β-cell gene expression. As the LIM homeodomain protein ISL1 is a suggested Wnt pathway downstream target, we hypothesize that it mediates metabolic functions of TCF7L2. We aimed to determine the role of ISL1 in mediating the function of TCF7L2 and the incretin hormone GLP-1 in pancreatic β-cells. The effect of dominant negative TCF7L2 (TCF7L2DN) mediated Wnt pathway functional knockdown on Isl1 expression was determined in βTCFDN mouse islets and in the rat insulinoma cell line INS-1 832/13. Luciferase reporter assay and chromatin immunoprecipitation were utilized to determine whether Isl1 is a direct downstream target of Tcf7l2 TCF7L2DN adenovirus infection and siRNA-mediated Isl1 knockdown on β-cell gene expression were compared. Furthermore, Isl1 knockdown on GLP-1 stimulated β-catenin S675 phosphorylation and insulin secretion was determined. We found that TCF7L2DN repressed ISL1 levels in βTCFDN islets and the INS-1 832/13 cell line. Wnt stimulators enhanced Isl1 promoter activity and binding of TCF7L2 on Isl1 promoter. TCF7L2DN adenovirus infection and Isl1 knockdown generated similar repression on expression of β-cell genes, including the ones that encode GLUT2 and GLP-1 receptor. Either TCF7L2DN adenovirus infection or Isl1 knockdown attenuated GLP-1-stimulated β-catenin S675 phosphorylation in INS-1 832/13 cells or mouse islets and GLP-1 stimulated insulin secretion in INS-1 832/13 or MIN6 cells. Our observations support the existence of TCF7L2-ISL1 transcriptional network, and we suggest that this network also mediates β-cell function of GLP-1.

Biphasic Electrical Stimulation Promotes Insulin Release

Biphasic (anodal pulse followed immediately by a cathodal pulse) electrical stimulation (10Hz, 5 ms duration) of rat insulinoma (Ins‐1) cells resulted in a 2–3‐fold increase in insulin release over 30 min that was stimulation period‐ and voltage‐dependent. Electrical stimulation of tissue culture medium (in the absence of cells) did not increase insulin release when subsequently added to cells, confirming that the insulin released by electrical stimulation was not due to substances generated by the culture medium. Electrical stimulation‐induced insulin release occurred in the absence or presence of glucose. Calcium channel blockade (using nifedipine or verapamil) inhibited basal insulin secretion and insulin secretion evoked by electrical stimulation, demonstrating that electrical stimulation‐induced insulin release involved activation of voltage‐dependent calcium channels and was not a non‐specific effect of stimulation on the plasma membrane. Basal insulin secretion and the increase in insulin release induced by electrical stimulation were also inhibited by the mitochondrial poisons antimycin A or oligomycin A, suggesting a role for mitochondrial function in insulin secretion. Electrical stimulation of cells also resulted in a more pronounced uptake of MitoTracker Red by mitochondria, indicating a more negative inner mitochondrial membrane potential. The failure of cells to accumulate the fluorogenic dye Cytotox green confirmed that electrical stimulation was not significantly disrupting the plasma membrane or inducing overt toxicity to the cells during the period of stimulation. These results provide further evidence that biphasic electrical stimulation can exert significant biological effects and elicit insulin release in a manner that is dependent upon mitochondrial activity and calcium channel activation but independent of glucose.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Liquiritigenin prevents palmitate-induced beta-cell apoptosis via estrogen receptor-mediated AKT activation

Liquiritigenin (LQ) is a major active component of licorice root, which is a flavone used for treating many diseases, including diabetes. LQ has been shown to exhibit a glucose-lowering effect in diabetic mice. Therefore, we investigated the potential of LQ to protect against lipotoxicity-induced beta-cell apoptosis and the underlying molecular mechanisms. Exposure of INS-1 rat insulinoma cells to LQ significantly increased cell viability and blocked palmitate (PA)-induced apoptosis, as evidenced by the reduction of Annexin-V-stained cells, cleaved caspase-3 levels, and poly (ADP-ribose) polymerase (PARP) activity, as well as upregulation of Bcl-2 expression. Moreover, LQ treatment significantly reduced the endoplasmic reticulum (ER) stress response by reducing phosphorylated protein kinase RNA-like endoplasmic reticulum kinase (PERK), phosphorylated eIF-2a, and CHOP expression in PA-treated INS-1 cells. The anti-apoptotic effect of LQ treatment was reversed through co-treatment with fulvestrant, a specific inhibitor of the estrogen receptor. LQ also increased AKT phosphorylation, and inactivation of this molecular event failed to decrease PERK phosphorylation with LQ treatment in PA-treated INS-1 cells. This effect was further accompanied by an inability to recover cell viability. These results suggest that LQ protects INS-1 cells from lipotoxicity-induced apoptosis by suppressing ER stress. We conclude that estrogen receptor-mediated AKT phosphorylation is one of the mechanisms contributing to the anti-apoptotic effect of LQ.