Cytokine-mediated Β-cell Death Research Articles

GHRH agonist MR-409 protects β-cells from streptozotocin-induced diabetes

Patients with type 1 diabetes (T1D) suffer from insufficient functional β-cell mass, which results from infiltration of inflammatory cells and cytokine-mediated β-cell death. Previous studies demonstrated the beneficial effects of agonists of growth hormone-releasing hormone receptor (GHRH-R), such as MR-409 on preconditioning of islets in a transplantation model. However, the therapeutic potential and protective mechanisms of GHRH-R agonists on models of T1D diabetes have not been explored. Using in vitro and invivo models of T1D, we assessed the protective propertie of the GHRH agonist, MR409 on β-cells. The treatment of insulinoma cell lines and rodent and human islets with MR-409 induces Akt signaling by induction of insulin receptor substrate 2 (IRS2), a master regulator of survival and growth in β-cells, in a PKA-dependent manner. The increase in cAMP/PKA/CREB/IRS2 axis by MR409 was associated with decrease in β-cell death and improved insulin secretory function in mouse and human islets exposed to proinflammatory cytokines. The assessment of the effects of GHRH agonist MR-409 in a model of T1D induced by low-dose streptozotocin showed that mice treated with MR-409 exhibited better glucose homeostasis, higher insulin levels, and preservation of β-cell mass. Increased IRS2 expression in β-cells in the group treated with MR-409 corroborated the invitro data and provided evidence for the underlying mechanism responsible for beneficial effects of MR-409 invivo. Collectively, our data show that MR-409 is a novel therapeutic agent for the prevention and treatment of β-cells death in T1D.

16-OR: Protein Kinase C d Mediates Cytokine-Induced ß-Cell Death via Upregulation of Pro-apoptotic Signaling

In type 1 diabetes (T1D) , autoreactive immune cells secrete cytokines, such as tumor necrosis factor-α (TNF-α) , interleukin-1β (IL-1β) , and interferon-γ (IFN-γ) that can initiate β-cell apoptosis. Protein kinase C delta (PKCδ) plays a role in mediating cytokine-induced β-cell apoptosis; however, the exact mechanisms and role in T1D are unknown. We hypothesize that PKCδ mediates cytokine-induced β-cell apoptosis via nuclear translocation and activation of pro-apoptotic signaling. Islets from WT or mice with a β-cell specific knockout of PKCδ (PKCδβ-KO) were cultured for 24hr with cytokines (10ng/ml TNFα, 5ng/ml IL-1β, 100ng/ml IFN-γ) , a caspase-3 inhibitor (Z-DEVD-FMK, 10µg/ml) , or an adenoviral vector for GFP-tagged PKCδ, GFP only, and a PKCδ mutant that cannot be cleaved by caspase-3 (CM-GFP-PKCδ) . Human islets were treated with 10μM PKCδ inhibitor (δV1-1) for 24hr with or without cytokines. Translocation was determined by GFP colocalization with a nuclear stain. Apoptosis was determined by fluorescent microscopy. PKCδ activity was determined with a FRET-based sensor in MIN6 cells. PKCδ’s role in activating pro-apoptotic signaling was determined via western blot. Decreasing PKCδ activity protected against cytokine-induced apoptosis in mouse (p=0.003) and human (p=0.020) islets. PKCδ translocation and activity increased in nuclei of cytokine treated islets (p<0.001, p=0.039) compared to untreated controls. Islets treated with caspase inhibitor (p<0.001) and CM-GFP-PKCδ islets were protected from cytokine-induced death. Western blot analysis demonstrates that PKCδ regulates pro-apoptotic signaling. Inhibiting PKCδ protected islets from cytokine-induced death. The role we identified for PKCδ in mediating pro-apoptotic proteins will provide novel insight to protect against cytokine-induced β-cell death. Our data suggests targeted inhibition of PKCδ has the potential to prevent cytokine-mediated β-cell death in T1D. Disclosure J.Collins: None. N.L.Farnsworth: None. R.K.Benninger: n/a. Funding American Diabetes Association (7-21-JDF-020) ; Juvenile Diabetes Research Foundation (1-FAC-2020-891-A-N)

84-OR: Laminin-Islet Interactions Protect against Cytokine-Mediated Beta-Cell Death via Down-Regulation of Protein Kinase C Delta

Islets are surrounded by a specialized extracellular matrix (ECM). Interactions with the peri-islet ECM, specifically laminin, promote islet viability and function. In T1D during immune infiltration the peri-islet ECM is broken down and β-cell death occurs in part through production of high levels of Th1 cytokines. Recent work has shown that cytokines alter islet function via protein kinase C Δ (PKCΔ) dependent mechanisms and that islet interactions with laminin reduce PKCΔ activity. We hypothesize that laminin-islet interactions protect against cytokine-mediated β-cell death via downregulation of PKCΔ. To test our hypothesis, we used a 3D reverse thermal gel (RTG) scaffold with either laminin-10 (RTG-Lam) or lysine (RTG-Lys) as a control. Islets were isolated from WT or mice with an inducible β-cell specific PKCΔ knockout (PKCΔ-βKO) and encapsulated in RTG-Lam and RTG-Lys for 24hrs with or without cytokines (10ng/mL TNF-α, 5ng/mL IL-1β, 100ng/mL IFN-γ). Viability, PKCΔ translocation, and PKCΔ activity were measured in encapsulated islets in situ using fluorescent biosensors or by western blot. PKCΔ-βKO islets were protected against cytokine-induced death compared to controls (p=0.003). Cytokines increased β-cell death in RTG-Lys (p<0.001), while islets encapsulated in RTG-Lam were protected against cytokine-mediated death (p<0.04). Islets encapsulated in RTG-Lam had reduced PKCΔ activity at the cell membrane compared to RTG-Lys and were also protected against cytokine-mediated increases in PKCΔ activity at the cell membrane. In summary, islet interactions with laminin protect against cytokine-induced β-cell death via downregulation of PKCΔ activity at the cell membrane. Our results suggest that loss of islet interactions with laminin, as occurs in T1D, may make islets more susceptible to cytokine-mediated death. This mechanism of β-cell death provides a novel therapeutic target to prevent β-cell death and halt the progression of T1D. Disclosure N.L. Farnsworth: None. R.A. Piscopio: None. R.K. Benninger: None. Funding JDRF (3-APF-2019-749-A-N, 1-FAC-2020-891-A-N); Colorado Clinical and Translational Sciences Institute (CO-M-19-133)

Exendin-4 overcomes cytokine-induced decreases in gap junction coupling via protein kinase A and Epac2 in mouse and human islets.

The pancreatic islets of Langerhans maintain glucose homeostasis through insulin secretion, where insulin secretion dynamics are regulated by intracellular Ca2+ signalling and electrical coupling of the insulin producing β-cells in the islet. We have previously shown that cytokines decrease β-cell coupling and that compounds which increase cAMP can increase coupling. In both mouse and human islets exendin-4, which increases cAMP, protected against cytokine-induced decreases in coupling and in mouse islets preserved glucose-stimulated calcium signalling by increasing connexin36 gap junction levels on the plasma membrane. Our data indicate that protein kinase A regulates β-cell coupling through a fast mechanism, such as channel gating or membrane organization, while Epac2 regulates slower mechanisms of regulation, such as gap junction turnover. Increases in β-cell coupling with exendin-4 may protect against cytokine-mediated β-cell death as well as preserve insulin secretion dynamics during the development of diabetes. The pancreatic islets of Langerhans maintain glucose homeostasis. Insulin secretion from islet β-cells is driven by glucose metabolism, depolarization of the cell membrane and an influx of calcium, which initiates the release of insulin. Gap junctions composed of connexin36 (Cx36) electrically couple β-cells, regulating calcium signalling and insulin secretion dynamics. Cx36 coupling is decreased in pre-diabetic mice, suggesting a role for altered coupling in diabetes. Our previous work has shown that pro-inflammatory cytokines decrease Cx36 coupling and that compounds which increase cAMP can increase Cx36 coupling. The goal of this study was to determine if exendin-4, which increases cAMP, can protect against cytokine-induced decreases in Cx36 coupling and altered islet function. In both mouse and human islets, exendin-4 protected against cytokine-induced decreases in coupling and preserved glucose-stimulated calcium signalling. Exendin-4 also protected against protein kinase Cδ-mediated decreases in Cx36 coupling. Exendin-4 preserved coupling in mouse islets by preserving Cx36 levels on the plasma membrane. Exendin-4 regulated Cx36 coupling via both protein kinase A (PKA)- and Epac2-mediated mechanisms in cytokine-treated islets. In mouse islets, modulating Epac2 had a greater impact in mediating Cx36 coupling, while in human islets modulating PKA had a greater impact on Cx36 coupling. Our data indicate that PKA regulates Cx36 coupling through a fast mechanism, such as channel gating, while Epac2 regulates slower mechanisms of regulation, such as Cx36 turnover in the membrane. Increases in Cx36 coupling with exendin-4 may protect against cytokine-mediated β-cell dysfunction to insulin secretion dynamics during the development of diabetes.

The No-Go and Nonsense-Mediated RNA Decay Pathways Are Regulated by Inflammatory Cytokines in Insulin-Producing Cells and Human Islets and Determine β-Cell Insulin Biosynthesis and Survival.

Stress-related changes in β-cell mRNA levels result from a balance between gene transcription and mRNA decay. The regulation of RNA decay pathways has not been investigated in pancreatic β-cells. We found that no-go and nonsense-mediated RNA decay pathway components (RDPCs) and exoribonuclease complexes were expressed in INS-1 cells and human islets. Pelo, Dcp2, Dis3L2, Upf2, and Smg1/5/6/7 were upregulated by inflammatory cytokines in INS-1 cells under conditions where central β-cell mRNAs were downregulated. These changes in RDPC mRNA or corresponding protein levels were largely confirmed in INS-1 cells and rat/human islets. Cytokine-induced upregulation of Pelo, Xrn1, Dis3L2, Upf2, and Smg1/6 was reduced by inducible nitric oxide synthase inhibition, as were endoplasmic reticulum (ER) stress, inhibition of Ins1/2 mRNA, and accumulated insulin secretion. Reactive oxygen species inhibition or iron chelation did not affect RDPC expression. Pelo or Xrn1 knockdown (KD) aggravated, whereas Smg6 KD ameliorated, cytokine-induced INS-1 cell death without affecting ER stress; both increased insulin biosynthesis and medium accumulation but not glucose-stimulated insulin secretion in cytokine-exposed INS-1 cells. In conclusion, RDPCs are regulated by inflammatory stress in β-cells. RDPC KD improved insulin biosynthesis, likely by preventing Ins1/2 mRNA clearance. Pelo/Xrn1 KD aggravated, but Smg6 KD ameliorated, cytokine-mediated β-cell death, possibly through prevention of proapoptotic and antiapoptotic mRNA degradation, respectively.

Do β-Cells Generate Peroxynitrite in Response to Cytokine Treatment?

The purpose of this study was to determine the reactive species that is responsible for cytokine-mediated β-cell death. Inhibitors of inducible nitric oxide synthase prevent this death, and addition of exogenous nitric oxide using donors induces β-cell death. The reaction of nitric oxide with superoxide results in the generation of peroxynitrite, and this powerful oxidant has been suggested to be the mediator of β-cell death in response to cytokine treatment. Recently, coumarin-7-boronate has been developed as a probe for the selective detection of peroxynitrite. Using this reagent, we show that addition of the NADPH oxidase activator phorbol 12-myristate 13-acetate to nitric oxide-producing macrophages results in peroxynitrite generation. Using a similar approach, we demonstrate that cytokines fail to stimulate peroxynitrite generation by rat islets and insulinoma cells, either with or without phorbol 12-myristate 13-acetate treatment. When forced to produce superoxide using redox cyclers, this generation is associated with protection from nitric oxide toxicity. These findings indicate that: (i) nitric oxide is the likely mediator of the toxic effects of cytokines, (ii) β-cells do not produce peroxynitrite in response to cytokines, and (iii) when forced to produce superoxide, the scavenging of nitric oxide by superoxide is associated with protection of β-cells from nitric oxide-mediated toxicity.

Epoxypukalide Induces Proliferation and Protects against Cytokine-Mediated Apoptosis in Primary Cultures of Pancreatic β-Cells

There is an urgency to find new treatments for the devastating epidemic of diabetes. Pancreatic β-cells viability and function are impaired in the two most common forms of diabetes, type 1 and type 2. Regeneration of pancreatic β-cells has been proposed as a potential therapy for diabetes. In a preliminary study, we screened a collection of marine products for β-cell proliferation. One unique compound (epoxypukalide) showed capability to induce β-cell replication in the cell line INS1 832/13 and in primary rat cell cultures. Epoxypukalide was used to study β-cell proliferation by [3H]thymidine incorporation and BrdU incorporation followed by BrdU/insulin staining in primary cultures of rat islets. AKT and ERK1/2 signalling pathways were analyzed. Cell cycle activators, cyclin D2 and cyclin E, were detected by western-blot. Apoptosis was studied by TUNEL and cleaved caspase 3. β-cell function was measured by glucose-stimulated insulin secretion. Epoxypukalide induced 2.5-fold increase in β-cell proliferation; this effect was mediated by activation of ERK1/2 signalling pathway and upregulation of the cell cycle activators, cyclin D2 and cyclin E. Interestingly, epoxypukalide showed protection from basal (40% lower versus control) and cytokine-induced apoptosis (80% lower versus control). Finally, epoxypukalide did not impair β-cell function when measured by glucose-stimulated insulin secretion. In conclusion, epoxypukalide induces β-cell proliferation and protects against basal and cytokine-mediated β-cell death in primary cultures of rat islets. These findings may be translated into new treatments for diabetes.

In Vitro Phenotypic, Genomic and Proteomic Characterization of a Cytokine-Resistant Murine β-TC3 Cell Line

Type 1 diabetes mellitus (T1DM) is caused by the selective destruction of insulin-producing β-cells. This process is mediated by cells of the immune system through release of nitric oxide, free radicals and pro-inflammatory cytokines, which induce a complex network of intracellular signalling cascades, eventually affecting the expression of genes involved in β-cell survival.The aim of our study was to investigate possible mechanisms of resistance to cytokine-induced β-cell death. To this purpose, we created a cytokine-resistant β-cell line (β-TC3R) by chronically treating the β-TC3 murine insulinoma cell line with IL-1β + IFN-γ. β-TC3R cells exhibited higher proliferation rate and resistance to cytokine-mediated cell death in comparison to the parental line. Interestingly, they maintained expression of β-cell specific markers, such as PDX1, NKX6.1, GLUT2 and insulin. The analysis of the secretory function showed that β-TC3R cells have impaired glucose-induced c-peptide release, which however was only moderately reduced after incubation with KCl and tolbutamide. Gene expression analysis showed that β-TC3R cells were characterized by downregulation of IL-1β and IFN-γ receptors and upregulation of SOCS3, the classical negative regulator of cytokines signaling. Comparative proteomic analysis showed specific upregulation of 35 proteins, mainly involved in cell death, stress response and folding. Among them, SUMO4, a negative feedback regulator in NF-kB and JAK/STAT signaling pathways, resulted hyper-expressed. Silencing of SUMO4 was able to restore sensitivity to cytokine-induced cell death in β-TC3R cells, suggesting it may play a key role in acquired cytokine resistance by blocking JAK/STAT and NF-kB lethal signaling.In conclusion, our study represents the first extensive proteomic characterization of a murine cytokine-resistant β-cell line, which might represent a useful tool for studying the mechanisms involved in resistance to cytokine-mediated β-cell death. This knowledge may be of potential benefit for patients with T1DM. In particular, SUMO4 could be used as a therapeutical target.

Involvement of SIRT1 in Zn2+, Streptozotocin, Non-Obese Diabetic, and Cytokine-Mediated Toxicities of β-cells.

To determine if the sirtuin pathway is involved in Zn2+-, streptozotocin-, or cytokine-mediated β-cell death in vitro, and streptozotocin-, or NOD induced T1DM in vivo. Sensitivity of MIN6 cells expressing empty vector, sirtuin protein-1 (SIRT1) or its siRNA, to Zn2+, streptozotocin, or cytokines, and effects on NAD+ levels were determined. Covariance of manipulating SIRT1 levels with diabetic incidence was tested in vivo. 1) sirtuin pathway inhibition or SIRT1 knockdown attenuated Zn2+-, STZ-, and cytokine-mediated toxicity and NAD+ loss in β-cells, 2) SIRT1 overexpression potentiated these toxicities, 3) young SIRT1 β-cell transgenic mice have improved glucose tolerance under basal conditions, but upon aging showed increased sensitivity to streptozotocin compared to SIRT1 +/- mice, and 4) SIRT1 +/- mice in an NOD background or exposed to streptozotocin trended toward reduced diabetic incidence and mortality compared to wildtype. These results have implicated SIRT1-mediated NAD+ loss in Zn2+, STZ, or cytokine toxicities of MIN6, and in NOD or streptozotocin T1DM animal models. Modulation of β-cell Zn2+ and NAD+ levels, and the sirtuin pathway could be novel therapeutic targets for T1DM.

Peripheral and Islet Interleukin-17 Pathway Activation Characterizes Human Autoimmune Diabetes and Promotes Cytokine-Mediated β-Cell Death

OBJECTIVECD4 T-cells secreting interleukin (IL)-17 are implicated in several human autoimmune diseases, but their role in type 1 diabetes has not been defined. To address the relevance of such cells, we examined IL-17 secretion in response to β-cell autoantigens, IL-17A gene expression in islets, and the potential functional consequences of IL-17 release for β-cells.RESEARCH DESIGN AND METHODSPeripheral blood CD4 T-cell responses to β-cell autoantigens (proinsulin, insulinoma-associated protein, and GAD65 peptides) were measured by IL-17 enzyme-linked immunospot assay in patients with new-onset type 1 diabetes (n = 50). mRNA expression of IL-17A and IFNG pathway genes was studied by qRT-PCR using islets obtained from subjects who died 5 days and 10 years after diagnosis of disease, respectively, and from matched control subjects. IL-17 effects on the function of human islets, rat β-cells, and the rat insulinoma cell line INS-1E were examined.RESULTSA total of 27 patients (54%) showed IL-17 reactivity to one or more β-cell peptides versus 3 of 30 (10%) control subjects (P = 0.0001). In a single case examined close to diagnosis, islet expression of IL17A, RORC, and IL22 was detected. It is noteworthy that we show that IL-17 mediates significant and reproducible enhancement of IL-1β/interferon (IFN)-γ–induced and tumor necrosis factor (TNF)-α/IFN-γ–induced apoptosis in human islets, rat β-cells, and INS-1E cells, in association with significant upregulation of β-cell IL17RA expression via activation of the transcription factors STAT1 and nuclear factor (NF)-κB.CONCLUSIONSCirculating IL-17+ β-cell–specific autoreactive CD4 T-cells are a feature of type 1 diabetes diagnosis. We disclose a novel pathway to β-cell death involving IL-17 and STAT1 and NF-κB, rendering this cytokine a novel disease biomarker and potential therapeutic target.

Cytokine toxicity in insulin-producing cells is mediated by nitro-oxidative stress-induced hydroxyl radical formation in mitochondria

Although nitric oxide (NO) and oxidative stress both contribute to proinflammatory cytokine toxicity in pancreatic β-cells during type 1 diabetes mellitus (T1DM) development, the interactions between NO and reactive oxygen species (ROS) in cytokine-mediated β-cell death have not been clarified. Exposure of insulin-producing RINm5F cells to IL-1β generated NO, while exposure to a combination of IL-1β, TNF-α, and IFN-γ, which simulates T1DM conditions, generated both NO and ROS. In theory, two reactions between NO and ROS are possible, one with the superoxide radical yielding peroxynitrite, and the other with hydrogen peroxide (H(2)O(2)) yielding hydroxyl radicals. Results of the present work exclude peroxynitrite involvement in cytokine toxicity to β-cells because its generation did not correlate with the toxic action of cytokines. On the other hand, we show that H(2)O(2), produced upon exposure of insulin-producing cell clones and primary rat islet cells to cytokines almost exclusively in the mitochondria, reacted in the presence of trace metal (Fe(++)) with NO forming highly toxic hydroxyl radicals, thus explaining the severe toxicity that causes apoptotic β-cell death. Expression of the H(2)O(2)-inactivating enzyme catalase in mitochondria protected against cytokine toxicity by preventing hydroxyl radical formation. We therefore conclude that proinflammatory cytokine-mediated β-cell death is due to nitro-oxidative stress-mediated hydroxyl radical formation in the mitochondria.