Johan Saldeen

Cytokine-induced apoptosis and necrosis are preceded by disruption of the mitochondrial membrane potential (Δψm) in pancreatic RINm5F cells: prevention by Bcl-2

The mechanisms of cytokine-induced beta-cell death are poorly characterised. In rat insulin-producing RINm5F cells, the combination of interleukin-1beta, interferon-gamma and tumour necrosis factor-alpha presently induced disruption of the mitochondrial membrane potential (Deltapsi(m)) as demonstrated by reduced JC-1 fluorescence. The reduction of Deltapsi(m) was maximal after 8 h and was preceded by increased formation of reactive oxygen species (ROS), as assessed by dichlorofluorescein-diacetate (DCFH-DA) fluorescence. A nitric oxide synthase-, but not a ROS-inhibitor, prevented cytokine-induced loss of Deltapsi(m). Overexpression of the anti-apoptotic protein Bcl-2 increased both JC-1 and DCFH-DA fluorescence, which was paralleled by protection against cytokine-induced apoptosis and necrosis. It is concluded that cytokines induce a nitric oxide-dependent disruption of Deltapsi(m) and that this may be a necessary event for both beta-cell apoptosis and necrosis. Bcl-2 may prevent beta-cell death by counteracting mitochondrial permeability transition.

Role of p38 mitogen-activated protein kinase (p38 MAPK) in cytokine-induced rat islet cell apoptosis

The signaling pathways mediating nitric oxide production and apoptosis in pancreatic beta-cells are not fully understood. We investigated cytokine-induced protein phosphorylation events in insulin-producing cells and evaluated their role in inducible nitric oxide synthase (iNOS) induction and cell death. Interleukin-1beta (IL-1beta), but not interferon-gamma (IFN-gamma), induced phosphorylation of p38 mitogen-activated protein kinase, c-Jun NH2-terminal kinase, and mitogen- and stress-activated protein kinase 1 (MSK1) in rat insulin-producing RINm5F cells. This was paralleled by an increased phosphorylation of the transcription factors activating transcription factor-2 (ATF-2) and cAMP-responsive element-binding protein (CREB). The p38 inhibitor SB203580 prevented cytokine-induced phosphorylation of CREB and MSK1, but not of ATF-2. IFN-gamma induced the phosphorylation of signal transducer and activator of transcription 1. The combination of IL-1beta and IFN-gamma increased both apoptosis and necrosis in rat islet cells. SB203580, but not the extracellular signal-regulated kinase inhibitor PD98059, partially prevented cytokine-induced apoptosis, an effect that was not associated with reduced nitrite production or lowered iNOS expression. In conclusion, cytokine-induced p38 activation participates in beta-cell apoptosis, possibly by a nitric oxide-independent mechanism or by enhancing the sensitivity to nitric oxide.

Efficient Gene Transfer to Dispersed Human Pancreatic Islet Cells In Vitro Using Adenovirus-Polylysine/DNA Complexes or Polycationic Liposomes

The establishment of gene delivery systems that result in efficient transfection of the pancreatic β-cells may generate an important tool for the study of IDDM and may also represent one critical step toward a clinical application of gene transfer for the prevention or early treatment of the disease. Using the reporter gene vectors pCAT and pCMV β-gal, we have investigated the efficiency of transfection mediated by calcium phosphate precipitation, the monocationic liposome Lipofectin, the polycationic liposome Lipofectamine, and adenovirus-polylysine (AdpL) DNA complexes in human, mouse, rat, and fetal porcine islet cells. In all species studied, calcium phosphate–mediated transfection resulted in lower chloramphenicol acetyl transferase (CAT) activities than the other methods. Intact human, mouse, and rat islets were poorly transfected by Lipofectin, Lipofectamine, and AdpL. When dispersed by trypsin treatment, however, human, mouse, rat, and fetal pig islet cells were efficiently transfected by Lipofectamine. Moreover, transfection of dispersed human and mouse islet cells using AdpL also resulted in high CAT activities. The percentage of cells staining positively for β-galactosidase after transfection with Lipofectamine was 49% for mouse, 56% for rat, and 57% for dispersed human islet cells. Transfection of human islet cells using AdpL, however, yielded 70% βgal–positive cells. Fluorescence-activated cell sorting-purified rat islet α- and β-cells were transfected with similar efficiency using Lipofectamine. CAT expression in human islet cells transfected with either Lipofectamine or AdpL reached a peak value after 5–7 days, followed by a gradual decline. It is concluded that transfection with AdpL or Lipofectamine are both efficient means to achieve transient expression of gene constructs in human and mouse islet cells, while for rat and fetal porcine islet cells, Lipofectamine is the most efficient of the agents investigated in this study.

Interleukin-1β induces the expression of HSP70, heme oxygenase and Mn-SOD in FACS-purified rat islet β-cells, but not in α-cells

Abstract The cytokine IL-1β has previously been demonstrated to induce the expression of the stress genes iNOS, hsp70, heme oxygenase and Mn-SOD in rat pancreatic islets in vitro. The aim of this study was to determine whether the IL-1β-induced effects are specific for the insulin producing β-cell, or whether other islet cells, such as the glucagon-producing α-cell, respond to IL-1β addition. Purified rat α- and β-cell suspensions were obtained by fluorescence-activated cell sorting and incubated with or without IL-1β (25 U/ml) for 24 h. The α- and β-cell contents of hsp70, heme oxygenase and Mn-SOD and medium nitrite levels were determined. It was found that IL-1β exposure induced the production of nitric oxide in β-cells, but not in α-cells. Moreover, the expression of hsp70, heme oxygenase and Mn-SOD was also induced in β-cells, but not in α-cells. There were no detectable levels of hsp70 in α-cells. It is concluded that the stress gene response following IL-1β exposure in markedly different in α- and β-cells. This finding may be of importance for the understanding of the autoimmune destruction of β-cells in insulin-dependent diabetes mellitus.

Nicotinamide-induced apoptosis in insulin producing cells is associated with cleavage of poly(ADP-ribose) polymerase

We have presently determined the effect of inhibition of the DNA repair enzyme poly(ADP-ribose) polymerase (PARP) on the occurrence of apoptosis in insulin-producing cells. The ADP-ribosylation activities of intact cells were decreased by incubation of RINm5F cells for 16 h with the PARP inhibitors nicotinamide (NA) (20-50 mM) or 3-aminobenzamide (3-ABA) (10 mM). Exposure to 20-50 mM NA or 10 mM 3-ABA both resulted in massive apoptosis in RINm5F cells. A 24 h exposure to 50 mM nicotinamide induced apoptosis in fetal but not adult rat islet cells. In addition, exposure of RINm5F cells to 50 mM NA for 12-24 h induced the appearance of the 85 kDa proteolytic PARP fragment, indicating activation of the ICE-like protease caspase-3. Incubation with 20-50 mM NA did not induce any consistent effects upon transcription factor NF-kappaB activity, demonstrating that this pathway is not involved in induction of apoptosis by NA. It is concluded that in insulin-producing cells with a high mitotic rate, inhibition of ADP-ribosylation--and consequently of auto-modification and release of PARP bound to DNA strand breaks--leads to activation of programmed cell death.

Nicotinamide- and caspase-mediated inhibition of poly(ADP-ribose) polymerase are associated with p53-independent cell cycle (G2) arrest and apoptosis.

Poly(ADP-ribose) polymerase (PARP), which is activated by DNA strand breaks, is involved in DNA repair and replication but, during apoptosis, undergoes early caspase-mediated cleavage. Activation of programmed cell death in response to DNA damage may rely on functional p53 protein. Tumor cells are commonly deficient in this oncogene product resulting in resistance to many cytostatic drugs. Here we report that nicotinamide-induced inhibition of poly(ADP-ribosyl)ation and cytokine-induced nitric oxide production both result in a transient increase in p53 levels in pancreatic tumor RINm5F cells. These treatments also induce disruption of the mitochondrial membrane potential (ΔΨm), as revealed using the mitochondrial probe JC-1, followed by PARP cleavage and apoptosis all of which are inhibited by the anti-apoptotic protein Bcl-2. Moreover, PARP-inhibition by nicotinamide or 3-aminobenzamide induces apoptosis and/or cell cycle arrest at the G2 checkpoint in all of four tested tumor cell lines of both mesenchymal and epithelial origin including mouse NIH-3T3 cells and p53 deficient human HeLa and Jurkat cells. Bcl-2 counteracts cytokine-, but not nicotinamide-induced G2 arrest. These findings indicate that both chemical and caspase-mediated inhibition of PARP activity, possibly by interfering with DNA replication and repair, may promote a p53-independent G2 arrest and apoptosis.

Novel experimental strategies to prevent the development of type 1 diabetes mellitus.

Abstract Type 1 diabetes is an autoimmune disease leading to extensive destruction of the pancreatic β-cells. Our research focusses on the role of β-cells during the course of the disease, aiming at finding novel strategies to enhance β-cell resistance against the cytotoxic damage inflicted by the immune system. Special attention has been paid to the possibility that cytokines released by the immune cells infiltrating the pancreatic islets can directly suppress and kill β-cells. Certain cytokines (interleukin-1β, tumor necrosis factor-α and interferon-γ) either alone or in combination, are able to activate signal transduction pathways in β-cells leading to transcription factor activation and de novo gene expression. In this context, it has been found that induction of inducible nitric oxide synthase mediates an elevated production of nitric oxide, which impairs mitochondrial function and causes DNA damage eventually leading to apoptosis and necrosis. However, other induced proteins SUCH AS heat shock protein 70 and superoxide dismutase may reflect a defense reaction elicited in the β-cells by the cytokines. Our strategy is to further seek for proteins involved in both destruction and protection of β-cells. Based on this knowledge, we plan to apply gene therapeutic approaches to increase expression of protective genes in β-cells. If this is feasible we will then evaluate the function and survival of such modified β-cells in animal models of type 1 diabetes such as the NOD mouse. The long-term goal for this research line is to find novel approaches to influence β-cell resistance in humans at risk of developing type 1 diabetes.

The Acid Sphingomyelinase Inhibitor SR33557 Counteracts TNF-α-Mediated Potentiation of IL-1β-Induced NF-κB Activation in the Insulin-Producing Cell Line RINm5F

Cytokines induce nitric oxide (NO) production and cell death in insulin-producing cells in vitro but the signaling pathways mediating the cytokine effects are not well characterized. The aim of this study was to determine whether sphingomyelinase (SMase) participates in cytokine signaling leading to NF- B activation, iNOS induction and cell death in insulin-producing cells. Acute exposure to IL-1β or TNF-α did not affect SMase activities in rat insulinoma (RINm5F) cells. TNF-α activated NF-B in gel shift experiments without inducing iNOS - as assessed by nitrite formation - whereas IL-1β stimulated both NF-B activation and iNOS induction. Natural ceramide did not activate NF-B or iNOS. However, both cera-mide and TNF-α potentiated IL-1β- induced activation of NF-B and iNOS. Moreover, the potentiating effects of TNF-α were counteracted by the acid SMase inhibitor SR33557. The combination of IL-1β and IFN-γ induced apoptosis in RINm5F cells, which was paralleled by a modest increase in acid SMase, whereas ceramide mainly induced necrosis. It is concluded that cytokine-induced p-cell signaling is associated with the induction of iNOS but not with enhanced SMase activities. However, TNF-α-mediated potentiation of the IL-1 β effect may involve an increased sensitivity to basal acid SMase activity. An increased acid SMase activity may participate in the execution of cytokine-induced P-cell apoptosis

ISLET EXPRESSION OF PERFORIN, FAS/APO-1 AND INTERLEUKIN-1 CONVERTING ENZYME (ICE) IN NON-OBESE DIABETIC (NOD) MICE

The aim of the present study was to correlate the islet expression of the apoptosis-associated factors Fas/Apo-1, FasL, ICE and perforin with the progression of beta-cell destruction in non-obese diabetic (NOD) mice. For this purpose, thymus and isolated pancreatic islets from male and female NOD mice of 5 and 15 weeks of age were subjected to immunoblot analysis. Islet expression of the Fas/Apo-1 receptor and ICE were increased in islets from female mice 15 weeks of age as compared to corresponding males. No Fas/Apo-1 or ICE signal was observed in the 5-week-old mice. The expression of perforin increased both in islets and in thymus with age and female gender. Islet expression of FasL could not be detected. Culture of isolated islets from NMRI mice in the presence of interleukin-1beta (IL-1beta) induced the expression of ICE. The present results support a direct role of the Fas/FasL and the perforin systems in the autoimmune destruction of insulin producing cells [corrected].

The SHB Adapter Protein Is Required for Normal Maturation of Mesoderm during in Vitro Differentiation of Embryonic Stem Cells

Definitive mesoderm arises from a bipotent mesendodermal population, and to study processes controlling its development at this stage, embryonic stem (ES) cells can be employed. SHB (Src homology 2 protein in β-cells) is an adapter protein previously found to be involved in ES cell differentiation to mesoderm. To further study the role of SHB in this context, we have established ES cell lines deficient for one (SHB+/-) or both SHB alleles (SHB-/-). Differentiating embryoid bodies (EBs) derived from these ES cell lines were used for gene expression analysis. Alternatively, EBs were stained for the blood vessel marker CD31. For hematopoietic differentiation, EBs were differentiated in methylcellulose. SHB-/- EBs exhibited delayed down-regulation of the early mesodermal marker Brachyury. Later mesodermal markers relatively specific for the hematopoietic, vascular, and cardiac lineages were expressed at lower levels on day 6 or 8 of differentiation in EBs lacking SHB. The expression of vascular endothelial growth factor receptor-2 and fibroblast growth factor receptor-1 was also reduced in SHB-/- EBs. SHB-/- EBs demonstrated impaired blood vessel formation after vascular endothelial growth factor stimulation. In addition, the SHB-/- ES cells formed fewer blood cell colonies than SHB+/+ ES cells. It is concluded that SHB is required for appropriate hematopoietic and vascular differentiation and that delayed down-regulation of Brachyury expression may play a role in this context.