Dariush Mokhtari

Amelioration of diabetes by imatinib mesylate (Gleevec): role of beta-cell NF-kappaB activation and anti-apoptotic preconditioning

It was recently reported that tyrosine kinase inhibitor imatinib mesylate (Gleevec®) improves Type 2 diabetes, possibly by decreasing insulin resistance. However, as both Type 2 and Type 1 diabetes are characterized by β‐cell dysfunction and death, we investigated whether imatinib counteracts diabetes by maintaining β‐cell function. We observed that imatinib counteracted diabetes in two animal models, the streptozotocin‐injected mouse and the nonobese diabetes mouse, and that this was paralleled by a partial preservation of the β‐cell mass. In addition, imatinib decreased the death of human β‐cells in vitro when exposed to NO, cytokines, and streptozotocin. The imatinib effect was mimicked by siRNA‐mediated knockdown of c‐Abl mRNA. Imatinib enhanced β‐cell survival by promoting a state similar to ischemic preconditioning, as evidenced by NF‐B activation, increased NO and reactive oxygen species production, and depolarization of the inner mitochondrial membrane. Imatinib did not suppress islet cell death in the presence of an NF‐B inhibitor, suggesting that NF‐B activation is a necessary step in the antiapoptotic action of imatinib. We conclude that imatinib mediates β‐cell survival and that this could contribute to the beneficial effects observed in diabetes.—Hägerkvist, R., Sandler, S., Mokhtari, D., Welsh, N. Amelioration of diabetes by imatinib mesylate (Gleevec®): role of β‐cell NF‐B activation and anti‐apoptotic preconditioning. FASEB J. 21, 618–628 (2007)

Thrombospondin-1: An Islet Endothelial Cell Signal of Importance for β-Cell Function

OBJECTIVE Loss of thrombospondin (TSP)-1 in pancreatic islets has been shown to cause islet hyperplasia. This study tested the hypothesis that endothelial-derived TSP-1 is important for β-cell function. RESEARCH DESIGN AND METHODS Islet function was evaluated both in vivo and in vitro. Messenger RNA and protein expression were measured by real-time PCR and Western blot, respectively. The role of endothelial-derived TSP-1 for β-cell function was determined using a transplantation design in which recipient blood vessels either were allowed to grow or not into the transplanted islets. RESULTS TSP-1–deficient mice were glucose intolerant, despite having an increased β-cell mass. Moreover, their islets had decreased glucose-stimulated insulin release, (pro)insulin biosynthesis, and glucose oxidation rate, as well as increased expression of uncoupling protein-2 and lactate dehydrogenase-A when compared with control islets. Almost all TSP-1 in normal islets were found to be derived from the endothelium. Transplantation of free and encapsulated neonatal wild-type and TSP-1–deficient islets was performed in order to selectively reconstitute with TSP-1–positive or –negative blood vessels in the islets and supported that the β-cell defects occurring in TSP-1–deficient islets reflected postnatal loss of the glycoprotein in the islet endothelial cells. Treatment of neonatal TSP-1–deficient mice with the transforming growth factor (TGF)β-1–activating sequence of TSP-1 showed that reconstitution of TGFβ-1 activation prevented the development of decreased glucose tolerance in these mice. Thus, endothelial-derived TSP-1 activates islet TGFβ-1 of importance for β-cells. CONCLUSIONS Our study indicates a novel role for endothelial cells as functional paracrine support for pancreatic β-cells.

Potential utility of small tyrosine kinase inhibitors in the treatment of diabetes

Altered tyrosine kinase signalling has been implicated in several diseases, paving the way for the development of small-molecule TKIs (tyrosine kinase inhibitors). TKIs such as imatinib, sunitinib and dasatinib are clinically used for treating chronic myeloid leukaemia, gastrointestinal stromal tumours and other malignancies. In addition to their use as anti-cancer agents, increasing evidence points towards an anti-diabetic effect of these TKIs. Imatinib and other TKIs counteract diabetes not only in non-obese diabetic mice, but also in streptozotocin diabetic mice, db/db mice, high-fat-treated rats and humans with T2D (Type 2 diabetes). Although the mechanisms of protection need to be investigated further, the effects of imatinib and other TKIs in human T2D and the rapidly growing findings from animal models of T1D (Type 1 diabetes) and T2D are encouraging and give hope to improved treatment of human diabetes. In the present article, we review the anti-diabetic effects of TKIs which appear to involve both protection against beta-cell death and improved insulin sensitivity. Considering the relatively mild side effects of TKIs, we hypothesize that TKIs could be used to treat new-onset T1D, prevent T1D in individuals at high risk of developing the disease, treat the late stages of T2D and improve the outcome of islet transplantation.

Overexpression of the nuclear factor-κB subunit c-Rel protects against human islet cell death in vitro.

The transcription factor nuclear factor (NF)-κB is known to modulate rates of apoptosis and may therefore play a role in the increased β-cell death that occurs in type 1 and type 2 diabetes. The aim of the present investigation was to study the expression of NF-κB subunits in human islet cells and whether overexpression of the NF-κB subunit c-Rel affects islet cell survival. We detected expression of p65, Rel-B, p50, p105, p52, and the ribosomal protein S3 (rpS3) in human islet cells. Among these, only p65 and rpS3 were translocated from the cytosolic to the nuclear fraction in response to cytokines. Interestingly, rpS3 participated in p65 binding to the κB-element in gel shift analysis experiments. We observed cytoplasmic c-Rel expression in vivo in 6J mice, and signs of nuclear translocation in β-cells of infiltrated nonobese diabetic islets. Human islet cells were also dispersed by trypsin treatment and transduced with a c-Rel adenoviral vector. This resulted in increased expression of c-Rel and inhibitory factor κB, increased κB-binding activity, and augmented protein levels of Bcl-X(L,) c-IAP2, and heat shock protein 72. c-Rel expression in human islet cells protected against cytokine-induced caspase 3 activation and cell death. c-Rel protected also against streptozotocin- and H(2)O(2)-induced cell death, in both intact rat islets and human islet cells. We conclude that rpS3 participates in NF-κB signaling and that a genetic increase in the activity of the NF-κB subunit c-Rel results in protection against cell death in human islets.

The MAPK Kinase Kinase-1 Is Essential for Stress-Induced Pancreatic Islet Cell Death

The aim of the present investigation was to characterize the role of the MAPK kinase kinase-1 (MEKK-1) in stress-induced cell death of insulin producing cells. We observed that transient overexpression of the wild type MEKK-1 protein in the insulin-producing cell lines RIN-5AH and betaTC-6 increased c-Jun N-terminal kinase (JNK) phosphorylation and augmented cell death induced by diethylenetriamine/nitroso-1-propylhydrazino)-1-propanamine (DETA/NO), streptozotocin (STZ), and hydrogen peroxide (H2O2). Furthermore, DETA/NO or STZ induced a rapid threonine phosphorylation of MEKK-1. Silencing of MEKK-1 gene expression in betaTC-6 and human dispersed islet cells, using in vitro-generated diced small interfering RNA, resulted in protection from DETA/NO, STZ, H2O2, and tunicamycin induced cell death. Moreover, in DETA/NO-treated cells diced small interfering RNA-mediated down-regulation of MEKK-1 resulted in decreased activation of JNK but not p38 and ERK. Inhibition of JNK by treatment with SP600125 partially protected against DETA/NO- or STZ-induced cell death. In summary, our results support an essential role for MEKK-1 in JNK activation and stress-induced beta-cell death. Increased understanding of the signaling pathways that augment or diminish beta-cell MEKK-1 activity may aid in the generation of novel therapeutic strategies in the treatment of type 1 diabetes.

Imatinib mesilate-induced phosphatidylinositol 3-kinase signalling and improved survival in insulin-producing cells: role of Src homology 2-containing inositol 5′-phosphatase interaction with c-Abl

Aims/hypothesisIt is not clear how small tyrosine kinase inhibitors, such as imatinib mesilate, protect against diabetes and beta cell death. The aim of this study was to determine whether imatinib, as compared with the non-cAbl-inhibitor sunitinib, affects pro-survival signalling events in the phosphatidylinositol 3-kinase (PI3K) pathway.MethodsHuman EndoC-βH1 cells, murine beta TC-6 cells and human pancreatic islets were used for immunoblot analysis of insulin receptor substrate (IRS)-1, Akt and extracellular signal-regulated kinase (ERK) phosphorylation. Phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3] plasma membrane concentrations were assessed in EndoC-βH1 and MIN6 cells using evanescent wave microscopy. Src homology 2-containing inositol 5′-phosphatase 2 (SHIP2) tyrosine phosphorylation and phosphatase and tensin homologue deleted on chromosome 10 (PTEN) serine phosphorylation, as well as c-Abl co-localisation with SHIP2, were studied in HEK293 and EndoC-βH1 cells by immunoprecipitation and immunoblot analysis. Gene expression was assessed using RT-PCR. Cell viability was measured using vital staining.ResultsImatinib stimulated ERK(thr202/tyr204) phosphorylation in a c-Abl-dependent manner. Imatinib, but not sunitinib, also stimulated IRS-1(tyr612), Akt(ser473) and Akt(thr308) phosphorylation. This effect was paralleled by oscillatory bursts in plasma membrane PI(3,4,5)P3 levels. Wortmannin induced a decrease in PI(3,4,5)P3 levels, which was slower in imatinib-treated cells than in control cells, indicating an effect on PI(3,4,5)P3-degrading enzymes. In line with this, imatinib decreased the phosphorylation of SHIP2 but not of PTEN. c-Abl co-immunoprecipitated with SHIP2 and its binding to SHIP2 was largely reduced by imatinib but not by sunitinib. Imatinib increased total β-catenin levels and cell viability, whereas sunitinib exerted negative effects on cell viability.Conclusions/interpretationImatinib inhibition of c-Abl in beta cells decreases SHIP2 activity, which results in enhanced signalling downstream of PI3 kinase.

siRNA produced by recombinant dicer mediates efficient gene silencing in islet cells.

Abstract: RNA interference (RNAi) is emerging as a powerful and convenient tool for studying gene function and genetic variation. RNAi is mediated by 21‐ to 23‐nucleotide‐long, small interfering RNAs (siRNA) produced from larger double‐stranded RNAs in vivo by the RNase III family enzyme Dicer. To overcome the problems associated with the use of predesigned synthetic siRNA molecules, a novel method utilizing the in vitro activity of recombinant Dicer has been developed recently. In nonislet cells, it has been demonstrated that a pool of siRNA, generated by Dicer from in vitro transcribed dsRNA (d‐siRNA), mediates convenient, efficient, and reproducible gene silencing in various cell types. The aim of this study was to evaluate the ability of d‐siRNA to silence endogenous gene expression in pancreatic islet cells. We observed that liposomal transfection mediates efficient transport of siRNA in up to 90% of dispersed islet cells and that d‐siRNA mediates almost complete and nontoxic silencing of an endogenous mRNA, the messenger coding for the nonreceptor tyrosine kinase c‐Abl. The approach described here using d‐siRNA provides an important tool for elucidating gene function in further studies of pancreatic islets and diabetes pathophysiology.

MAPK Kinase Kinase-1 Is Essential for Cytokine-Induced c-Jun NH2-Terminal Kinase and Nuclear Factor-κB Activation in Human Pancreatic Islet Cells

OBJECTIVE—The transcription factor nuclear factor-κB (NF-κB) and the mitogen-activated protein kinases (MAPKs) c-Jun NH2-terminal kinase (JNK) 1/2 are known to play decisive roles in cytokine-induced damage of rodent β-cells. The upstream events by which these factors are activated in response to cytokines are, however, uncharacterized. The aim of the present investigation was to elucidate a putative role of the MAPK kinase kinase-1 (MEKK-1) in cytokine-induced signaling. RESEARCH DESIGN AND METHODS—To establish a functional role of MEKK-1, the effects of transient MEKK-1 overexpression in βTC-6 cells, achieved by lipofection and cell sorting, and MEKK-1 downregulation in βTC-6 cells and human islet cells, achieved by diced–small interfering RNA treatment, were studied. RESULTS—We observed that overexpression of wild-type MEKK-1, but not of a kinase dead MEKK-1 mutant, resulted in potentiation of cytokine-induced JNK activation, inhibitor of κB (IκB) degradation, and cell death. Downregulation of MEKK-1 in human islet cells provoked opposite effects, i.e., attenuation of cytokine-induced JNK and MKK4 activation, IκB stability, and a less pronounced NF-κB translocation. βTC-6 cells with a downregulated MEKK-1 expression displayed also a weaker cytokine-induced iNOS expression and lower cell death rates. Also primary mouse islet cells with downregulated MEKK-1 expression were protected against cytokine-induced cell death. CONCLUSIONS—MEKK-1 mediates cytokine-induced JNK- and NF-κB activation, and this event is necessary for iNOS expression and cell death.

Effects of Imatinib Mesylate (Gleevec) on human islet NF-kappaB activation and chemokine production in vitro.

Purpose Imatinib Mesylate (Gleevec) is a drug that potently counteracts diabetes both in humans and in animal models for human diabetes. We have previously reported that this compound in human pancreatic islets stimulates NF-κB signaling and islet cell survival. The aim of this study was to investigate control of NF-κB post-translational modifications exerted by Imatinib and whether any such effects are associated with altered islet gene expression and chemokine production in vitro. Procedures Human islets were either left untreated or treated with Imatinib for different timepoints. IκB-α and NF-κB p65 phosphorylation and methylation were assessed by immunoblot analysis. Islet gene expression was assessed using a commercial Pathway Finder microarray kit and RT-PCR. Islet chemokine production was determined by flow cytometric bead array analysis. Findings Human islet IκB-α and Ser276-p65 phosphorylation were increased by a 20 minute Imatinib exposure. Methylation of p65 at position Lys221 was increased after 60 min of Imatinib exposure and persisted for 3 hours. Microarray analysis of islets exposed to Imatinib for 4 hours revealed increased expression of the inflammatory genes IL-4R, TCF5, DR5, I-TRAF, I-CAM, HSP27 and IL-8. The islet release of IL-8 was augmented in islets cultured over night in the presence of Imatinib. Following 30 hours of Imatinib exposure, the cytokine-induced IκB-α and STAT1 phosphorylation was abolished and diminished, respectively. The cytokine-induced release of the chemokines MIG and IP10 was lower in islets exposed to Imatinib for 30 hours. Conclusion Imatinib by itself promotes a modest activation of NF-κB. However, a prolonged exposure of human islets to Imatinib is associated with a dampened response to cytokines. It is possible that Imatinib induces NF-κB preconditioning of islet cells leading to lowered cytokine sensitivity and a mitigated islet inflammation.

Impaired glucose homeostasis in Shb−/− mice

Src homology 2 domain-containing protein B (SHB) is an adapter protein involved in the regulation of beta-cell and endothelial cell function. We have recently obtained the Shb knockout mouse, and consequently, the aim of this study was to assess the effect of Shb deletion upon beta-cell function and blood glucose homeostasis. Shb-/- mice display an elevated basal blood glucose concentration, and this increase is maintained during insulin challenge in insulin sensitivity tests. To assess glucose-induced insulin secretion, pancreata were perfused, and it was observed that Shb-/- first phase insulin secretion was blunted during glucose stimulation. Gene expression of Shb-/- islets shortly after isolation was altered, with increased pancreatic and duodenal homeobox gene-1 (Pdx1) gene expression and reduced expression of Vegf-A. Islet culture normalized Pdx1 gene expression. The microvascular density of the Shb-/- islets was reduced, and islet capillary endothelial cell morphology was changed suggesting an altered microvascular function as a contributing cause to the impaired secretory activity. Capacitance measurements of depolarization-induced exocytosis indicate a direct effect on the exocytotic machinery, in particular a dramatic reduction in readily releasable granules, as responsible for the insulin-secretory defect operating in Shb-/- islets. Shb-/- mice exhibited no alteration of islet volume or beta-cell area. In conclusion, loss of Shb impairs insulin secretion, alters islet microvascular morphology, and increases the basal blood glucose concentration. The impaired insulin secretory response is a plausible underlying cause of the metabolic impairment observed in this mutant mouse.