Induce Beta-cell Apoptosis Research Articles

Synergistic effect of schizandrin A and DNase I knockdown on high glucose induced beta cell apoptosis by decreasing intracellular calcium concentration.

To explore the synergistic effect of deoxyribonuclease I (DNase I) knockdown combined with Schizandrin A (Sch A) in protecting islet beta-cells (β-cells) from apoptosis under high-glucose (HG) conditions. The concentration of Sch A was detected by Cell Counting Kit-8 (CCK-8). High glucose-cultured rat insulinoma beta cell line (RIN-M5F) cells were treated with Sch A and transfected with DNase I small interfering RNA (siRNA). Cell apoptosis rate and apoptosis-related protein level were examined by flow cytometry and Western blot method respectively. In addition, Na-K-adenosine triphosphatease (Na-K-ATPase) and Ca-Mg-ATPase activity, cell membrane potential, and intracellular Ca concentration was also examined respectively. Our study revealed that HG stimulation can cause a significant increase in DNase I level and cell apoptosis rate. However, Sch A combined with DNase I knockdown can significantly decrease the cell apoptosis rate and apoptosis-related protein levels such as BAX ( 0.05) and Caspase-3 ( 0.01). In addition, we also found that the combination of Sch A and DNase I knockdown can dramatically increase cell membrane potential level, Na-K-ATPase, and Ca-Mg-ATPase activity. Meanwhile, intracellular Ca concentration was also found to be significantly decreased by the synergistic effect of Sch A and DNase I knockdown. Overall, our study reveals a synergistic effect of Sch A and DNase I knockdown in protecting β-cells from HG-induced apoptosis.

TXNIP overexpression in mice enhances streptozotocin-induced diabetes severity

Thioredoxin-interacting protein (TXNIP) is a key player in the endocrine pancreas; it induces beta cell apoptosis, such that TXNIP deficiency promotes beta cell survival. To study its function in more detail, we generated transgenic mice with ubiquitous overexpression of TXNIP. CBATXNIP/+ mice were investigated under basal conditions and after being challenged in diet-induced obesity (DIO) and streptozotocin-induced type 1 diabetes mellitus (T1DM) models. TXNIP overexpression caused no effect in the DIO model, contrasting to the already reported TXNIP-deficient mice. However, in the T1DM background, CBATXNIP/+ animals showed significantly enhanced blood glucose and increased glucose levels in a glucose tolerance test. Finally, the beta cell mass of CBATXNIP/+ transgenic animals in the T1DM model was significantly reduced compared to control littermates. Our study demonstrates that overexpression of TXNIP doesn't affect blood glucose parameters under basal conditions. However, overexpression of TXNIP in a T1DM model enhances the severity of the disease.

369-OR: Defining the Role of RIPK3 in Beta Cells and the Mechanism of Its Activation by ER Stress

Receptor interacting protein kinase 3 (RIPK3) can activate and be activated by ER stress. Islet inflammation associated with underlying activation of RIPK3 is conserved from zebrafish to humans. However, the role of RIPK3 in mammalian beta cells and the mechanism by which ER stress activates RIPK3 are not well defined. To investigate the role of RIPK3 in beta cell function, we generated beta cell specific Ripk3 knockout mice (Ripk3ßKO) in C57BL/6J background with or without the Akita mutation (Ins2C96Y/+, which causes ER stress induced beta cell apoptosis and early onset diabetes) (Ripk3ßKOAkita) . We found that the glucose tolerance was worsened in Ripk3ßKO mice than wildtype controls after 16 weeks of high fat diet. Similarly, Ripk3ßKOAkita mice had higher random blood glucose, lower circulating insulin, and worse glucose tolerance than Akita C57BL/6J mice as early as 4 weeks of age. Unexpectedly, isolated islets from Ripk3ßKO and Ripk3ßKOAkita mice have comparable glucose stimulated insulin secretion (GSIS) and are more resilient to palmitate-induced lipotoxicity than their control counterparts. Similarly, pharmacological inhibition of RIPK3 protected human islets from palmitate-induced impairment of GSIS ex vivo. We are currently investigating the role of specific islet inflammation stages in the divergent GSIS phenotypes between in vivo and ex vivo. To investigate how ER stress activates RIPK3 in beta cells, we tested the effect of inhibiting classic UPR branches on RIPK3 activation in MIN6 cells, a mouse beta cell line. We found that IRE1a activation appears to play an essential role in RIPK3 activation. Interestingly, Ripk3 knockdown in MIN6 cells also decreased IRE1a levels. We next seek to define the mechanism underlying the crosstalk between RIPK3 and IRE1a signaling pathways. Disclosure L.Yang: None. B.Yang: None. X.Tong: None. C.Dai: None. Z.Tang: None. W.Chen: None. Funding National Institutes of Health (DK117147)

Alpk1 Sensitizes Pancreatic Beta Cells to Cytokine-Induced Apoptosis via Upregulating TNF-α Signaling Pathway.

Pancreatic beta cell failure is the hallmark of type 1 diabetes (T1D). Recent studies have suggested that pathogen recognizing receptors (PRRs) are involved in the survival, proliferation and function of pancreatic beta cells. So far, little is known about the role of alpha-protein kinase 1 (ALPK1), a newly identified cytosolic PRR specific for ADP-β-D-manno-heptose (ADP-heptose), in beta cell survival. In current study we aimed to fill the knowledge gap by investigating the role of Alpk1 in the apoptosis of MIN6 cells, a murine pancreatic beta cell line. We found that the expression of Alpk1 was significantly elevated in MIN6 cells exposed to pro-inflammatory cytokines, but not to streptozotocin, low-dose or high-dose glucose. Activation of Alpk1 by ADP heptose alone was insufficient to induce beta cell apoptosis. However, it significantly exacerbated cytokine-induced apoptosis in MIN6 cells. Mechanistic investigations showed that Alpk1 activation was potent to further induce the expression of tumor necrosis factor (TNF)-α and Fas after cytokine stimulation, possibly due to enhanced activation of the TIFA/TAK1/NF-κB signaling axis. Treatment of GLP-1 receptor agonist decreased the expression of TNF-α and Fas and improved the survival of beta cells exposed to pro-inflammatory cytokines and ADP heptose. In summary, our data suggest that Alpk1 sensitizes beta cells to cytokine-induced apoptosis by potentiating TNF-α signaling pathway, which may provide novel insight into beta cell failure and T1D development.

High glucose-induced PRDX3 acetylation contributes to glucotoxicity in pancreatic β-cells: Prevention by Teneligliptin

Chronic hyperglycemia has deleterious effects on pancreatic β-cell function and survival in type 2 diabetes (T2D) due to the low expression level of endogenous antioxidants in the β-cells. Peroxiredoxin-3 (PRDX3) is a mitochondria specific H202 scavenger and protects the cell from mitochondrial damage. However, nothing is known about how glucotoxicity influences PRDX3 function in the pancreatic beta cells. Exposure of rat insulinoma INS-1 cells and human beta cells (1.1B4) to high glucose conditions (30mM) stimulated acetylation of PRDX3 which facilitates its hyper-oxidation causing mitochondrial dysfunction by SIRT1 degradation. SIRT1 deficiency induces beta cell apoptosis via NOX-JNK-p66Shc signalosome activation. Herein we investigated the direct effect of Teneligliptin, a newer DPP-4 inhibitor on beta-cell function and survival in response to high glucose conditions. Teneligliptin treatment enhances SIRT1 protein levels and activity by USP22, an ubiquitin specific peptidase. Activated SIRT1 prevents high glucose-induced PRDX3 acetylation by SIRT3 resulted in inhibition of PRDX3 hyper-oxidation thereby strengthening the mitochondrial antioxidant defense. Notably, we identify PRDX3 as a novel SIRT3 target and show their physical interaction. Intriguingly, inhibition of SIRT1 activity by EX-527 or SIRT1 siRNA knockdown exacerbated the SIRT3 mediated PRDX3 deacetylation which leads to peroxiredoxin-3 hyper-oxidation and beta-cell apoptosis by the activation of NOX-JNK-p66Shc signalosome. Collectively, our results unveil a novel and first direct effect of high glucose on PRDX3 acetylation on beta-cell dysfunction by impaired antioxidant defense and SIRT1 mediated SIRT3-PRDX3 activation by Teneligliptin suppresses high glucose-mediated mitochondrial dysfunction.

Mesenchymal stem cell-derived exosomes protect beta cells against hypoxia-induced apoptosis via miR-21 by alleviating ER stress and inhibiting p38 MAPK phosphorylation

BackgroundHypoxia is a major cause of beta cell death and dysfunction after transplantation. The aim of this study was to investigate the effect of exosomes derived from mesenchymal stem cells (MSCs) on beta cells under hypoxic conditions and the potential underlying mechanisms.MethodsExosomes were isolated from the conditioned medium of human umbilical cord MSCs and identified by WB, NTA, and transmission electron microscopy. Beta cells (βTC-6) were cultured in serum-free medium in the presence or absence of exosomes under 2% oxygen conditions. Cell viability and apoptosis were analysed with a CCK-8 assay and a flow cytometry-based annexin V-FITC/PI apoptosis detection kit, respectively. Endoplasmic reticulum stress (ER stress) proteins and apoptosis-related proteins were detected by the WB method. MiRNAs contained in MSC exosomes were determined by Illumina HiSeq, and treatment with specific miRNA mimics or inhibitors of the most abundant miRNAs was used to reveal the underlying mechanism of exosomes.ResultsExosomes derived from MSC-conditioned culture medium were 40–100 nm in diameter and expressed the exosome markers CD9, CD63, CD81, HSP70, and Flotillin 1, as well as the MSC markers CD73, CD90, and CD105. Hypoxia significantly induced beta cell apoptosis, while MSC exosomes remarkably improved beta cell survival. The WB results showed that ER stress-related proteins, including GRP78, GRP94, p-eIF2α and CHOP, and the apoptosis-related proteins cleaved caspase 3 and PARP, were upregulated under hypoxic conditions but were inhibited by MSC exosomes. Moreover, the p38 MAPK signalling pathway was activated by hypoxia and was inhibited by MSC exosomes. The Illumina HiSeq results show that MSC exosomes were rich in miR-21, let-7 g, miR-1246, miR-381, and miR-100. After transfection with miRNA mimics, the viability of beta cells under hypoxia was increased significantly by miR-21 mimic, and the p38 MAPK and ER stress-related proteins in beta cells were downregulated. These changes were reversed after exosomes were pretreated with miR-21 inhibitor.ConclusionsExosomes derived from MSCs could protect beta cells against apoptosis induced by hypoxia, largely by carrying miR-21, alleviating ER stress and inhibiting p38 MAPK signalling. This result indicated that MSC exosomes might improve encapsulated islet survival and benefit diabetes patients.

TNFα-induced DLK activation contributes to apoptosis in the beta-cell line HIT.

Reduction in beta-cell mass and function contributes to the pathogenesis of diabetes mellitus type 2. The proinflammatory cytokines tumor necrosis factor (TNF)α and interleukin (IL)-1β have been implicated in the pathogenesis of this disease. Overexpression of the dual leucine zipper kinase (DLK) inhibits beta-cell function and induces apoptosis in the beta-cell line HIT. In the present study, it was investigated whether TNFα or IL-1β stimulates DLK enzymatic activity. Immunoblot analysis, transient transfection with luciferase reporter gene assays, and immunofluorescence were used. In contrast to IL-1β, TNFα stimulated DLK kinase activity, which was dependent on the c-Jun N-terminal kinase (JNK). Furthermore, DLK contributed to TNFα-induced JNK phosphorylation. The phosphorylation of DLK on Ser-302 within the activation loop was required for DLK to stimulate JNK and to inhibit CREB-dependent gene transcription. TNFα induced apoptosis in a time- and concentration-dependent manner and inhibited CREB-directed gene transcription in HIT cells. The reduction of endogenous DLK by small interfering or small hairpin RNA attenuated TNFα's effects on apoptosis and CREB-dependent transcription. These data suggest that TNFα induces beta-cell apoptosis through activation of DLK thereby inhibiting the beta-cell protective transcription factor CREB. Furthermore, activation of DLK by a well-known diabetic risk factor supports the role of DLK in the pathogenesis of diabetes mellitus. Thus, the inhibition of DLK might prevent or retard the pathogenesis of diabetes mellitus type 2.

Elevated systemic glutamic acid level in the non-obese diabetic mouse is Idd linked and induces beta cell apoptosis.

Although type 1 diabetes (T1D) is a T-cell-mediated disease in the effector stage, the mechanism behind the initial beta cell assault is less understood. Metabolomic differences, including elevated levels of glutamic acid, have been observed in patients with T1D before disease onset, as well as in pre-diabetic non-obese diabetic (NOD) mice. Increased levels of glutamic acid damage both neurons and beta cells, implying that this could contribute to the initial events of T1D pathogenesis. We investigated the underlying genetic factors and consequences of the increased levels of glutamic acid in NOD mice. Serum glutamic acid levels from a (NOD×B6)F2 cohort (n = 182) were measured. By genome-wide and Idd region targeted microsatellite mapping, genetic association was detected for six regions including Idd2, Idd4 and Idd22. In silico analysis of potential enzymes and transporters located in and around the mapped regions that are involved in glutamic acid metabolism consisted of alanine aminotransferase, glutamic-oxaloacetic transaminase, aldehyde dehydrogenase 18 family, alutamyl-prolyl-tRNA synthetase, glutamic acid transporters GLAST and EAAC1. Increased EAAC1 protein expression was observed in lysates from livers of NOD mice compared with B6 mice. Functional consequence of the elevated glutamic acid level in NOD mice was tested by culturing NOD. Rag2-/- Langerhans' islets with glutamic acid. Induction of apoptosis of the islets was detected upon glutamic acid challenge using TUNEL assay. Our results support the notion that a dysregulated metabolome could contribute to the initiation of T1D. We suggest that targeting of the increased glutamic acid in pre-diabetic patients could be used as a potential therapy.

The microRNA-200 family regulates pancreatic beta cell survival in type 2 diabetes.

Pancreatic beta cell death is a hallmark of type 1 (T1D) and type 2 (T2D) diabetes, but the molecular mechanisms underlying this aspect of diabetic pathology are poorly understood. Here we report that expression of the microRNA (miR)-200 family is strongly induced in islets of diabetic mice and that beta cell-specific overexpression of miR-200 in mice is sufficient to induce beta cell apoptosis and lethal T2D. Conversely, mir-200 ablation in mice reduces beta cell apoptosis and ameliorates T2D. We show that miR-200 negatively regulates a conserved anti-apoptotic and stress-resistance network that includes the essential beta cell chaperone Dnajc3 (also known as p58IPK) and the caspase inhibitor Xiap. We also observed that mir-200 dosage positively controls activation of the tumor suppressor Trp53 and thereby creates a pro-apoptotic gene-expression signature found in islets of diabetic mice. Consequently, miR-200-induced T2D is suppressed by interfering with the signaling of Trp53 and Bax, a proapoptotic member of the B cell lymphoma 2 protein family. Our results reveal a crucial role for the miR-200 family in beta cell survival and the pathophysiology of diabetes.

Direct regulation of mitogen-activated protein kinase kinase kinase DLK by calcineurin

Loss of beta-cell mass is the most important factor for the pathogenesis of type 1 and type 2 diabetes. Our previous results showed that inhibition of the calcium-calmodulin dependent phosphatase calcineurin by the structurally distinct immunosuppressant drugs cyclosporin A and tacrolimus decreased insulin gene transcription and both drugs stimulated the catalytic activity of the mitogen-activated protein kinase kinase kinase 12 (DLK Dual Leucine zipper kinase) and induced beta-cell apoptosis. In the present study, the regulation of DLK by calcineurin was investigated.

Role and Potential Therapeutic Use of TRAIL in Diabetes

TNF-related apoptosis-inducing ligand (TRAIL) is a TNF superfamily member, defined by its high homology to CD95L/FasL and TNF-alpha. It is known for its strong selective apoptotic effect on many transformed cell lines and tumor cells but not in most normal cell types. TRAIL appears to be a more complex molecule than predicted, with a higher therapeutic potential than previously anticipated. This is mainly because it has 5 different receptors that it can bind to in contrast to other TNF family members with one or two receptors; it is expressed widely in human tissues; and it has anti-inflammatory effects. For instance, type 1 diabetes (T1D) development was exacarbated in non-obese diabetic (NOD) mice when TRAIL function was blocked, and TRAIL -/- C57BL/6 mice developed T1D at a much earlier stage following streptozotocin (STZ) injection, compared to mice which displayed normal TRAIL expression. Furthermore, TRAIL displayed a pro-angiogenic effect in primary human vascular endothelial cells. It also induced survival against the apoptosis triggered by pro-inflammatory cytokines in vascular smooth muscle cells, as well as promoting migration and proliferation. Results from studies aiming to clear out the role of TRAIL in diabetes development suggest a protective role for this molecule on beta cells. TRAIL generally does not induce beta cell apoptosis, plays a protective role in these cells against the apoptotic effects of cytokines such as FasL and TNF-alpha, and its receptors are expressed at a significant level in the pancreatic islets. While TRAIL’s role or potential use in diabetes is investigated in various cell and animal systems by many reasearch groups including ours, it is still not clearly identified. Our previous and current results, which mainly support a protective role for TRAIL, will be discussed comparatively with similar and contradictory results of other groups.

PO271 THE PLASMA ADIPONECTIN LEVELS IN ELDERLY PATIENTS WITH PREDIABETES

Background: Ginseng have been proposed to mediate antidiabetic effect. The active components of ginseng are thought to be ginsenoside and distributed in many parts of the ginseng plant. Rg3, one of the active ingredients of ginseng saponins, has been known about the effects on molecularmechanism related to diabetes including inhibition the palmitate-induced beta-cell apoptosis and enhancement the glucose uptake. Glucose variability could induce a higher degree of apoptosis and decreased the insulin secretory capacity in beta-cells. In the present study, we investigated whether Rg3 suppresses intermittent high glucose(IHG)induced beta-cell apoptosis and improved insulin secretion in INS-1 pancreatic beta-cells. Method: To perform Glucose Fluctuation and protection effect by ginsenoside Rg3, mediums were aspirated and new medium containing Glucose Fluctuation or/and Rg3 concentrations was added and incubated for 2days. Annexin V staining for apoptosis detection analyzed by FACsort. Cell viability was determined by CCK-8 assay. The optical density was measured at 450 nm by automatic plate reader. Quantification of secreted insulin protein by ELISA was measured using Rat/mouse Insulin ELISA kit. BrdU staining was added in and quantification of secreted insulin proteins were analyzed by FACSort. Result: After 48hrs, INS-1 cell treated with alternative high glucose concentration(33mM glucose) and low glucose concentration(5mM) increased beta-cells apoptosis by 5.37 fold. But culturing with Rg3 1mM, 5mM,10mM, the percentages of apoptotic cells were reduced by dose dependently. Cell viability was reduced 63% by IHG. This toxic effect was also attenuated by culturing with Rg3. Additionally, when we added BrdU to culture medium and labeling of proliferating cells, cell proliferation was tended to be increased. And the ratio of quantification of secreted insulin protein/total protein was also tended to be increased with Rg3 dose dependently. Conclusion: Ginsenoside Rg3 suppresses intermittent high glucose-induced apoptosis and increases cell viability. Also it has tendancy to increase cell proliferation and the ratio of insulin secreting protein/total protein in INS-1 pancreatic beta-cells.

High glucose and free fatty acids induce beta cell apoptosis via autocrine effects of ADP acting on the P2Y13 receptor

While high levels of glucose and saturated fatty acids are known to have detrimental effects on beta cell function and survival, the signalling pathways mediating these effects are not entirely known. In a previous study, we found that ADP regulates beta cell insulin secretion and beta cell apoptosis. Using MIN6c4 cells as a model system, we investigated if autocrine/paracrine mechanisms of ADP and purinergic receptors are involved in this process. High glucose (16.7mmol/l) and palmitate (100μmol/l) rapidly and potently elevated the extracellular ATP levels, while mannitol was without effect. Both tolbutamide and diazoxide were without effect, while the calcium channel blocker nifedipine, the volume-regulated anion channels (VRAC) inhibitor NPPB, and the pannexin inhibitor carbenoxolone could inhibit both effects. Similarly, silencing the MDR1 gene also blocked nutrient-generated ATP release. These results indicate that calcium channels and VRAC might be involved in the ATP release mechanism. Furthermore, high glucose and palmitate inhibited cAMP production, reduced cell proliferation in MIN6c4 and increased activated Caspase-3 cells in mouse islets and in MIN6c4 cells. The P2Y(13)-specific antagonist MRS2211 antagonized all these effects. Further studies showed that blocking the P2Y(13) receptor resulted in enhanced CREB, Bad and IRS-1 phosphorylation, which are known to be involved in beta cell survival and insulin secretion. These findings provide further support for the concept that P2Y(13) plays an important role in beta cell apoptosis and suggest that autocrine/paracrine mechanisms, related to ADP and P2Y(13) receptors, contribute to glucolipotoxicity.

Reply to association between tamoxifen treatment and diabetes

We thank Drs. Hejazi and Rastmanesh for their interest in our article. The purpose of our study was to determine whether there is an association between tamoxifen therapy and diabetes, and our findings will need to be confirmed in other populations. Although our population-based databases have the advantage of providing a large sample size with which to address that question, they lack detailed clinical information such as that regarding weight gain and other risk factors. We agree that the mechanisms postulated for this association are uncertain. We acknowledge that tamoxifen is a selective estrogen receptor modulator, and that its effects on estrogen vary by tissue site. As we discuss in our article,1 there is evidence that estrogen protects against beta-cell failure and diabetes,2-4 leading to speculation that the observed increase in diabetes may be related to its estrogen inhibitory effects at the beta-cell level. This hypothesis is supported by the finding that tamoxifen induces beta-cell apoptosis and insulin deficiency in mice through direct estrogen antagonism.3 However, as the authors point out, alternative hypotheses such as the potential inflammatory effects of tamoxifen also need to be considered. Further studies that include more comprehensive clinical and metabolic data are needed to explore this intriguing finding. Supported by Cancer Care Ontario and the Ontario Institute for Cancer Research (through funding provided by the Ministry of Health and Long-Term Care and the Ministry of Research and Innovation of the Government of Ontario) and a Canadian Diabetes Association/ Canadian Institutes for Health Research (CIHR) Clinician Scientist Award. CONFLICT OF INTEREST DISCLOSURES Dr. Lipscombe receives salary support from the Canadian Diabetes Association/CIHR Clinician Scientist Award. Lorraine L. Lipscombe MD, MSc* , Paula A. Rochon MD, MPH* , * Women's College Research Institute, Women's College Hospital, Department of Medicine, University of Toronto; Institute for Clinical Evaluative Sciences, Department of Health Policy, Management and Evaluation, University of Toronto; Sunnybrook Health Sciences Centre, Toronto, Ontario, Canada

Deletion of Fas protects islet beta cells from cytotoxic effects of human islet amyloid polypeptide.

AIMS/HYPOTHESIS: Islet amyloid, which is mainly composed of human islet amyloid polypeptide (hIAPP), is a pathological characteristic of type 2 diabetes and also forms in cultured and transplanted islets. We used islet beta cells as well as two ex vivo models of islet amyloid formation, cultured human islets and hIAPP-expressing transgenic mouse islets with or without beta cell Fas deletion, to test whether: (1) the aggregation of endogenous hIAPP induces Fas upregulation in beta cells; and (2) deletion or blocking of Fas protects beta cells from amyloid toxicity. METHODS: INS-1, mouse or human islet cells were cultured with hIAPP alone, or with amyloid inhibitor or Fas antagonist. Non-transduced islets, and human islets or hIAPP-expressing mouse islets transduced with an adenovirus that delivers a human proIAPP-specific small interfering RNA (siRNA) (Ad-ProhIAPP-siRNA) were cultured to form amyloid. Mouse islets expressing hIAPP with or without Fas were similarly cultured. Beta cell Fas upregulation, caspase-3 activation, apoptosis and function, and islet IL-1β levels were assessed. RESULTS: hIAPP treatment induced Fas upregulation, caspase-3 activation and apoptosis in INS-1 and islet cells. The amyloid inhibitor or Fas antagonist reduced apoptosis in hIAPP-treated beta cells. Islet cells with Fas deletion had lower hIAPP-induced beta cell apoptosis than those expressing Fas. Ad-ProhIAPP-siRNA-mediated amyloid inhibition reduced Fas upregulation and IL-1β immunoreactivity in human and hIAPP-expressing mouse islets. Cultured hIAPP-expressing mouse islets with Fas deletion had similar amyloid levels, but lower caspase-3 activation and beta cell apoptosis, and a higher islet beta:alpha cell ratio and insulin response to glucose, compared with islets expressing Fas and hIAPP. CONCLUSIONS/INTERPRETATION: The aggregation of biosynthetic hIAPP produced in islets induces beta cell apoptosis, at least partially, via Fas upregulation and the Fas-mediated apoptotic pathway. Deletion of Fas protects islet beta cells from the cytotoxic effects of endogenously secreted (and exogenously applied) hIAPP.

GLP-1 Can Protect Proinflammatory Cytokines Induced Beta Cell Apoptosis through the Ubiquitination

Background: Proinflammatory cytokines are one of the causes of diabetes mellitus. However, the exact molecular mechanism by which proinflammatory cytokines induce β-cell death remains to be clearly elucidated. Glucagon-like peptide-1 (GLP-1) affects the stimulation of insulin secretion and the preservation of β-cells. Additionally, it may exert an antiapoptotic effect on β cells; however, the mechanism underlying this effect has yet to be demonstrated. Therefore, we investigated the protective effects of GLP-1 in endoplasmic reticulum (ER)-mediated β-cell apoptosis using proinflammatory cytokines. Methods: To induce ER stress, hamster insulin-secreting tumor (HIT)-T15 cells were treated using a mixture of cytokines. Apoptosis was evaluated via MTT assay, Hoechst 33342 staining, and annexin/propidium iodide (PI) flow cytometry. The mRNA and protein expression levels of ER stress-related molecules were determined via PCR and Western blotting, respectively. Nitric oxide was measured with Griess reagent. The levels of inducible nitric oxide synthase (iNOS) mRNA and protein were analyzed via real-time PCR and Western blot, respectively. iNOS protein degradation was evaluated via immunoprecipitation. We pretreated HIT-T15 cells with exendin (Ex)-4 for 1 hour prior to the induction of stress. Results: We determined that Ex-4 exerted a protective effect through nitric oxide and the modulation of ER stress-related molecules (glucose-regulated protein [GRP]78, GRP94, and CCAAT/enhancer-binding protein homologous protein [CHOP]) and that Ex-4 stimulates iNOS protein degradation via the ubiquitination pathway. Additionally, Ex-4 also induced the recovery of insulin2 mRNA expression in β cells. Conclusion: The results of this study indicate that GLP-1 may protect β cells against apoptosis through the ubiquitination pathway. (Endocrinol Metab 26:142-149, 2011)

Exendin‐4 protects pancreatic beta cells from human islet amyloid polypeptide‐induced cell damage: potential involvement of AKT and mitochondria biogenesis

Glucagon-like peptide-1 (GLP-1) stimulates beta-cell proliferation and enhances beta-cell survival, whereas oligomerization of human islet amyloid polypeptide (hIAPP) may induce beta-cell apoptosis and reduce beta-cell mass. Type 2 diabetes is associated with increased expression of IAPP. As GLP-1-based therapy is currently developed as a novel antidiabetic therapy, we examined the potential protective action of the GLP-1 receptor agonist exendin-4 on hIAPP-induced beta-cell apoptosis. The study was performed in clonal insulinoma (INS-1E) cells. Both method of transcriptional and translational and sulphorhodamine B (SRB) assays were used to evaluate cell viability and cell mass. Western blot analysis was applied to detect protein expression. Transfection of constitutively active protein kinase B (PKB/AKT) was performed to examine the role of AKT. Mitochondrial biogenesis was quantified by mitogreen staining and RT-PCR. First, we confirmed that hIAPP induced cell apoptosis and growth inhibition in INS-1E cells. These effects were partially protected by exendin-4 in association with partial recovery of the hIAPP-mediated AKT inhibition. Furthermore, AKT constitutive activation attenuated hIAPP-induced apoptosis, whereas PI3K/AKT inhibition abrogated exendin-4-mediated effects. These findings suggest that the antiapoptotic and proliferative effects of exendin-4 in hIAPP-treated INS-1E cells were partially mediated through AKT pathway. Moreover, hIAPP induced FOXO1 but inhibited pdx-1 nucleus translocation. These effects were restored by exendin-4. Finally, mitogreen staining and RT-PCR revealed enhanced mitochondrial biogenesis by exendin-4 treatment. Collectively, these results suggest that GLP-1 receptor agonist protects beta cells from hIAPP-induced cell death partially through the activation of AKT pathway and improved mitochondrial function.

Beta-cell selective K ATP-channel activation protects beta-cells and human islets from human islet amyloid polypeptide induced toxicity

Background and aims In type 2 diabetes mellitus (T2DM) chronic beta-cell stimulation and oligomers of aggregating human islet amyloid polypeptide (h-IAPP) cause beta-cell dysfunction and induce beta-cell apoptosis. Therefore we asked whether beta-cell rest prevents h-IAPP induced beta-cell apoptosis. Materials and methods We induced beta-cell rest with a beta-cell selective K ATP-channel opener (K ATPCO) in RIN cells and human islets exposed to h-IAPP versus r-IAPP. Apoptosis was quantified by time-lapse video microscopy (TLVM) in RIN cells and TUNEL staining in human islets. Whole islets were also studied with TLVM over 48 h to examine islet architecture. Results In RIN cells and human islets h-IAPP induced apoptosis (p < 0.001 h-IAPP versus r-IAPP). Concomitant incubation with K ATPCO inhibited apoptosis (p < 0.001). K ATPCO also reduced h-IAPP induced expansion of whole islets (disintegration of islet architecture) by ~ 70% (p < 0.05). Thioflavin-binding assays show that K ATPCO does not directly inhibit amyloid formation. Conclusions Opening of K ATP-channels reduces beta-cell vulnerability to apoptosis induced by h-IAPP oligomers. This effect is not due to a direct interaction of K ATPCO with h-IAPP, but might be mediated through hyperpolarization of the beta-cell membrane induced by opening of K ATP-channels. Induction of beta-cell rest with beta-cell selective K ATP-channel openers may provide a strategy to protect beta-cells from h-IAPP induced apoptosis and to prevent beta-cell deficiency in T2DM.

Sterol regulatory element‐binding protein‐1c knockdown protected INS‐1E cells from lipotoxicity

The reduction in insulin secretory capacity and beta-cell mass has been attributed, at least partially, to lipotoxicity, which may contribute to the development of type 2 diabetes. Chronic free fatty acids (FFA) exposure impairs pancreatic beta-cell function and induces beta-cell apoptosis. This study is to elucidate the underlying molecular mechanisms. We exposed INS-1E pancreatic beta-cell line to palmitate or oleate, and measured the glucose stimulated insulin secretion (GSIS). The effect of FFA on sterol regulatory element-binding protein (SREBP)-1c lipogenic pathway, and expression of genes involved in beta-cell functions, including AMPK (AMP-activated protein kinase), UCP-2 (uncoupling protein-2), IRS-2 (insulin receptor substrate-2), PDX-1 (pancreatic duodenal homeobox-1), GLUT-2 (glucose transporter-2) and B cell lymphoma/leukaemia-2 (Bcl-2) were investigated. Apoptosis of these exposed cells was determined by MitoCapture, Annexin V-Cy3 or terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay. Cell lipid accumulation was measured by oil red O staining or TG extraction. Also SREBP-1c expression knockdown were used. FFA treatment resulted in SREBP-1c overexpression, impaired GSIS, lipid accumulation, apoptosis of INS-1E cells. In addition, the expression of lipogenic genes and UCP-2 were upregulated, but AMPK, IRS-2, PDX-1, GLUT-2 and Bcl-2 were downregulated in the exposed cells. However, these lipotoxic effects of FFA were largely prevented by induction of a SREBP-1c small interfering RNA. These data suggest a strong correlation between FFA treatment and SREBP-1c activation in INS-1E cells. SREBP-1c might be a major factor responsible for beta-cell lipotoxicity, and SREBP-1c knockdown could protect INS-1E cells from lipotoxicity, which is implicating a therapeutic potential for treating diabetes related to lipotoxicity.

Angiotensin II exerts glucose-dependent effects on Kv currents in mouse pancreatic β-cells via angiotensin II type 2 receptors

Hyperglycemia-associated glucotoxicity induces beta-cell apoptosis but the underlying mechanisms are unknown. Interestingly, prolonged exposure to high glucose upregulates the expression and function of the renin-angiotensin system (RAS). We hypothesize that the voltage-gated outward potassium (K(v)) current, which governs beta-cell membrane potential and insulin secretion, has a role in glucotoxicity. In this study, we investigated the effects of prolonged exposure to high glucose on mouse pancreatic beta-cells and concurrent effects on the RAS by examining changes in expression of angiotensin II (ANG II) receptors and changes in the expression and activity of K(v) channels. beta-Cells were incubated in high glucose medium for 1-7 days and then were examined with electrophysiological and molecular biology techniques. Prolonged exposure to high glucose produced a marked increase in beta-cell primary K(v) channel subunit, K(v)2.1, expression and K(v) current amplitude. Enhanced expression of ANG II type 1 receptor (AT(1)R) was also observed under high glucose conditions, whereas blockade of AT(1)R by losartan did not alter K(v) channel expression. External application of ANG II reduced K(v) current amplitude under normal, but not high, glucose conditions. The effect of ANG II on K(v) channel gating was abolished by ANG II type 2 receptor (AT(2)R) antagonism. These data suggest that hyperglycemia alters beta-cell function through modification of the K(v) channel which may be associated with the RAS.