Rat Insulinoma Research Articles

Orexin-A Stimulates Insulin Secretion Through the Activation of the OX1 Receptor and Mammalian Target of Rapamycin in Rat Insulinoma Cells.

The study aimed to investigate the involvement of the mammalian target of rapamycin (mTOR) signaling pathway in orexin-A/OX1 receptor-induced insulin secretion in rat insulinoma INS-1 cells. Rat insulinoma INS-1 cells were grown and treated with various concentrations of orexin-A, with or without OX1 receptor-selective antagonist SB674042 or the phosphatidylinositol 3-kinase/mTOR antagonist PF-04691502. Insulin release experiments, Western blot analysis, and statistical analysis were conducted using INS-1 cells. Our results showed that treating cells with orexin-A increased the expression of the OX1 receptor and the phosphorylation of mTOR in a concentration-dependent manner. An increase in insulin secretion was also observed for cells treated with orexin-A. We further demonstrated that the increase in insulin secretion was dependent on the activation of the OX1 receptor and mTOR signaling pathway by using the OX1 receptor-selective antagonist SB674042 or the phosphatidylinositol 3-kinase/mTOR antagonist PF-04691502, which abolished the effects of orexin-A treatment. Our results concluded that orexin-A/OX1 receptor stimulates insulin secretion by activating AKT and its downstream target, mTOR. Therefore, orexins may regulate the energy balance for cell survival with the involvement of mTOR in this process.

1,25(OH)2D3 Activates Autophagy to Protect against Oxidative Damage of INS-1 Pancreatic Beta Cells.

Diabetes mellitus is a serious disease endangering human health worldwide. Vitamin D (Vit D) is a well-characterized regulator of calcium-phosphorus metabolism that also exerts other biological effects extending far beyond mineral homeostasis. Some epidemiological studies have suggested that Vit D has a role in defense against diabetes, although the mechanism remains unclear. Autophagy, an intracellular catabolic process, is necessary to maintain the normal structure and function of host cells. In our previous study, we found that Vit D could induce autophagy of pancreatic beta cells and prevent insulitis, although the underlying mechanisms remain to be fully elucidated. In this study, the protective effect of 1,25(OH)2D3, the physiologically active metabolite of Vit D, against streptozotocin-induced cytotoxicity in rat insulinoma cell line (INS-1) cells was explored. Cell viability and insulin secretion of INS-1 cells in response to different treatments were measured with a cell counting kit and enzyme-linked immune absorbent assay (ELISA), respectively. In addition, malondialdehyde (MDA) content and total antioxidant capacity (T-AOC) were measured by ELISA. RT-PCR and Western blot analyses were used to detect autophagy levels, reactive oxygen species (ROS) was assessed by fluorescence microscope, ultrastructure analysis was performed using transmission electron microscopy. The results demonstrated that 1,25(OH)2D3 could increase cell viability and insulin secretion of INS-1 cells, and protected cells from oxidative damage induced by streptozotocin (STZ) through autophagy activation. These findings shed light on mechanisms underlying the ameliorative effects of Vit D on diabetes mellitus.

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.

Candidate plasticity gene 16 mediates suppression of insulin gene expression in rat insulinoma INS-1 cells under glucotoxic conditions

Chronic hyperglycemia causes pancreatic β-cell dysfunction, impaired insulin secretion and suppression of insulin gene expression, referred to as glucotoxicity. Insulin gene expression is regulated by several protein kinases and protein phosphatases. However, the molecular mechanisms of the suppressed insulin gene expression in glucotoxicity are not fully understood. In this study, we employed rat insulinoma INS-1 cells as a model of pancreatic glucotoxicity. In INS-1 cells, insulin gene expression is up-regulated by incubation with 11.2 mM glucose for 7 days and down-regulated by incubation with 22.4 mM glucose for the same period. To identify the protein kinases and protein phosphatases involved in the suppression of insulin gene expression, we analyzed gene expression in INS-1 cells cultured with 11.2 mM or 22.4 mM glucose for 7 days using microarray analysis and real-time PCR. The expression levels of nine protein kinases were affected by glucotoxic conditions. In particular, CPG16 expression level was increased in INS-1 cells under these conditions. Transfection of CPG16 decreased insulin promoter activity, whereas kinase-dead mutant of CPG16 did not affect this. These results suggest that CPG16 plays a role in the suppression of insulin gene expression in pancreatic β-cells under glucotoxic conditions.

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.

INSULIN SECRETAGOGUE EFFECT OF ROOTS OF RAVENALA MADAGASCARIENSIS SONN. - AN IN VITRO STUDY

Objective: The objective of this study was to establish the cytotoxicity profile and to evaluate the insulin secretagogue effect of ethanolic root extract of Ravenala madagascariensis Sonn.
 Methods: The cell viability of rat insulinoma 5F (RIN5F) cell lines over the treatment of plant extract was assessed by 3-(4,5-dimethyl-2-thiazolyl)- 2,5-diphenyltetrazolium bromide assay. The insulin-releasing effect was evaluated by insulin secretion assay over RIN5F cell lines by enzyme-linked immunosorbent assay.
 Results: The ethanolic extract of the roots of R. madagascariensis Sonn. showed negligible cytotoxicity at 20–40 μg/ml, and hence, concentrations up to 40 μg/ml were used in insulin secretion assay. The ethanolic root extract at 20 and 40 μg/ml significantly (p<0.05 compared to control) stimulated the insulin release in a dose-dependent manner even in the presence of glucose at lower and higher concentrations (5 and 10 mM).
 Conclusion: Thus, our results validate its traditional claim in the treatment of diabetes by stimulating the secretion of insulin, thereby suggesting a possible mechanism of its antidiabetic effect.

Viscothionin purified from mistletoe (Viscum album var. coloratum Ohwi) induces insulin secretion from pancreatic beta cells

Ethnopharmacological relevanceMistletoe (Viscum album), an evergreen parasitic plant, has been widely used as an oriental phytomedicine to treat diabetes mellitus. However, it is unknown which mistletoe constituent exerts the beneficial effect against the disease. In this study, we examined the hypoglycemic activity of mistletoe and investigated whether the polypeptide viscothionin, purified from mistletoe, was responsible for the activity. Materials and methodsMistletoe extracts were prepared by heating mistletoe powder made of leaves and twigs in water for 3, 6, 9, and 12 h. Rat insulinoma RINm5F cells were used to test the cytotoxicity of the extracts and their effects on the secretion of insulin and its precursor, C-peptide. The inhibitory effects of a mistletoe extract on glucose absorption were measured using an α-glucosidase inhibition assay. To determine the component of mistletoe responsible for the observed effects, the mistletoe extract was precipitated with ethanol or hydrolyzed with a protease for further testing. A potential active constituent of mistletoe was isolated by chromatography and molecular weight cut-off fractionation, and its ability to induce insulin secretion was investigated. ResultsA 12-h heat-treated mistletoe extract, showing no cytotoxicity, significantly increased the secretion of insulin and C-peptide by RINm5F cells and enhanced the expression of glucose transporter type 4 (GLUT-4), insulin receptor substrate 1 (IRS-1), and protein kinase B (also known as AKT) in differentiated C2C12 cells. The extract also inhibited α-glucosidase activity. After ethanol precipitation, the extract showed much stronger effects on insulin- and C-peptide-secreting activities of cells, whereas the enzyme-hydrolyzed extract was less effective than the original extract, suggesting that the effect was mediated by a proteinaceous constituent of mistletoe. Subsequent analysis showed that viscothionin, a heat-stable 6-kDa polypeptide isolated from mistletoe, increased the level of insulin secretion by more than 20-fold compared to that induced by the extract. ConclusionsOur study indicates that the hypoglycemic effect of mistletoe is mediated by its insulinotropic action and α-glucosidase inhibitory activity, and the effect is due to viscothionin, one of the major bioactive constituents of mistletoe.

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.

Functional proteasome complex is required for turnover of islet amyloid polypeptide in pancreatic β-cells

Human islet amyloid polypeptide (hIAPP) is the principal constituent of amyloid deposits and toxic oligomers in the pancreatic islets. Together with hyperglycemia, hIAPP-derived oligomers and aggregates are important culprits in type 2 diabetes mellitus (T2DM). Here, we explored the role of the cell's main proteolytic complex, the proteasome, in hIAPP turnover in normal and stressed β-cells evoked by chronic hyperglycemia. Moderate inhibition (10-35%) of proteasome activity/function in cultured human islets by the proteasome inhibitor lactacystin enhanced intracellular accumulation of hIAPP. Unexpectedly, prolonged (>1 h) and marked (>50%) impairment of proteasome activity/function had a strong inhibitory effect on hIAPP transcription and secretion from normal and stressed β-cells. This negative compensatory feedback mechanism for controlling IAPP turnover was also observed in the lactacystin-treated rat insulinoma β-cell line (INS 832/13), demonstrating the presence of an evolutionarily conserved mechanism for IAPP production. In line with these in situ studies, our current ex vivo data showed that proteasome activity and hIAPP expression are also down-regulated in islets isolated from T2DM subjects. Gene expression and promoter activity studies demonstrated that the functional proteasome complex is required for efficient activation of the hIAPP promoter and for full expression of IAPP's essential transcription factor, FOXA2. ChIP studies revealed that promoter occupancy of FoxA2 at the rat IAPP promoter region is an important and limiting factor for amylin expression in proteasome-impaired murine cells. This study suggests a novel regulatory pathway in β-cells involving proteasome, FOXA2, and IAPP, which can be possibly targeted to regulate hIAPP levels and islet amyloidosis in T2DM.

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.