Both in Type 1 and Type 2 diabetes there is a deficit in the number and function of β-cells. The reasons for increased death and decreased regeneration and function of β-cells remain unknown. Therefore, our overall aim is to study signaling events that participate in β-cell death, function and regeneration. We currently focus on signaling via the non-receptor tyrosine kinase c-Abl, ROS production occurring via the NADPH-dependent oxidases (NOX) and the transcription factor ZBED6, which appears to control β-cell proliferation and function. For these purposes we are studying human β-cells (EndoC-betaH1 cells and human islets) and we utilize techniques such as RNA-seq, CHiP-seq, qPCR, immunoblot analysis, flow cytometry, viral vector-mediated transduction, siRNA and in vivo models.
Role of tyrosine kinases in ß-cell apoptosis and diabetes
It has recently been observed that patients suffering from both leukemia and diabetes were cured from not only leukemia, but also diabetes, when treated with the tyrosine kinase inhibitor Imatinib. (Veneri et al., N Engl J Med. 2005 352(10):1049-50). An anti-diabetic action of Imatinib in Type 2 diabetes is further supported by our recent observation that Imatinib counteracts high-fat diet induced insulin resistance and hyperglycemia in rats (Hägerkvist et al., Clinical Science, (Lond). 2008 114(1):65-71). Moreover, in a study from 2009, Imatinib was also observed to induce remission of diabetes in db/db mice, possibly via decreasing insulin resistance and increasing the ß-cell mass (Han et al., Diabetes. 2009 58(2):329-3). Thus, in both animal models and in Type 2 diabetes patients Imatinib seems to improve glycemic control, possibly via an insulin sensitizing effect.
Imatinib appears to prevent and reverse not only Type 2 diabetes, but also diabetes of animal models with a Type 1 diabetes resembling disease. We have shown that Imatinib protects against ß-cell death in vitro and prevents diabetes in NOD mice and in streptozotocin-diabetic mice, both models for human ß-cell destruction and Type 1 diabetes (Hagerkvist et al., FASEB J. 2007 Feb;21(2):618-28, Hagerkvist et al., Cell Biol Int. 2006 30(12):1013-7). More recently, it has been observed by others that both Imatinib and Sunitinib not only prevented, but also reversed new-onset diabetes in NOD mice (Louvet et al., Proc Natl Acad Sci U S A. 2008 105(48):18895-900). Thus, there exists proof-of-principle in animal models for an anti-diabetic effect of Imatinib and similar tyrosine kinase inhibitors, and that a limited treatment period will not only reverse diabetes, but also mediate long-term protection against re-precipitation of the disease. This has led us (Mokhtari and Welsh, Clin Sci (Lond). 2009 118(4):241-7) and other investigators to propose clinical trials in which Imatinib is given to new-onset Type 1 diabetes patients.
The work by others and us indicates that Imatinib counteracts diabetes via different molecular mechanisms (Figure 1).
Figure 1 Possible mechanisms for the anti-diabetic effects of imatinib. Imatinib is known to inhibit the tyrosine kinases c-Abl, PDGFR, c-Kit and DDR1/2. Most likely, imatinib-induced protection against diabetes is mediated not by one single pathway, but via different molecular mechanisms. ß-Cell survival is promoted by inhibition of c-Abl, which leads to decreased activation of the pro-apoptotic MAPK JNK and increased activation of the anti-apoptotic transcription factor NF-κB. c-Abl inhibition might also lead to a dampened ER-stress response, via JNK or other pathways. Inhibition of PDGFR could contribute to decreasing peripheral insulin resistance and inflammatory processes, thereby promoting ß-cell survival. Moreover, inhibition of c-Kit and DDR1/2 might also add to the anti-diabetic effects of imatinib, possibly by interfering with inflammatory responses.
It appears that the four known targets of Imatinib, c-Abl, PDGFR, c-Kit and DDR1/2, may all play a role in the pathogenesis of diabetes. C-Abl is a proapoptotic tyrosine kinase that promotes ß-cell death when activated. Improper activation of the PDGF receptor has also been reported to occur in diabetes, and this may lead to increased insulin resistance of peripheral tissues. Activation of c-Kit and DDR1/2 is known to affect innate immunity, a component of the immune system that promotes inflammation and ß-cell dysfunction. Thus, it is conceivable that Imatinib, by targeting several pathways simultaneously, mediates a stronger antidiabetic effect than other drugs that affect only one particular pathway.
It is the aim of this project to elucidate closer the mechanisms by which tyrosine kinases control ß-cell death and function. We are currently investigating Imatinib-mediated control of NF-κB, JNK, p38, PI3-kinase, SHIP2, PTEN, FAK, IRS1/2, ß-catenin, AKT and ERK signaling events. For this purpose insulin producing cells, either at basal conditions or under stress, are analyzed by immunoprecipitation, immunoblotting, confocal microscopy, real-time PCR, microarray analysis, flow cytometry and gel shift analysis. Cells are also genetically manipulated by lentiviral vectors to achieve up-or down-regulation of specific gene products. Signaling events will be correlated to ß-cell survival and function, as assessed by analysis of insulin production and apoptotic events. This will hopefully lead to a better understanding of the molecular events by which Imatinib protects against diabetes. Such improved knowledge may pave the way for a novel and improved treatment of diabetes.
Role of ROS producing NADPH-dependent oxidases (NOX) in β-cell dysfunction
Loss of pancreatic islet function is a central hallmark in the pathogenesis of T2DM. In addition, it may be that also β-cell loss occurs in T2DM, and that this starts, after an initial phase of hyperinsulinemia, relatively late in the progression of the disease. The mechanisms resulting in beta cell failure in T2DM are not clear, but accumulating evidence point to a central role of oxidative stress as a result of overproduction of reactive oxygen species (ROS) (Figure 1).
The excessive production and accumulation of ROS is, at least in part, due to hyperactivity of the NADPH oxidases (NOX). The NOX family consists of seven isoforms (NOX1-5 and DUOX1-2), which perform normal cellular functions at basal conditions, but when persistently activated produce harmful levels of ROS. Hyperactivity of some of the isoforms has been found to be an important driver in a number of diseases including diabetes and diabetes complications . The present project will explore ways to protect against β-cell oxidative stress and deterioration by inhibiting NOX, and to define T2DM patient groups that would particularly benefit from treatment with such inhibitors. Novel NOX inhibitors are available to us via Glucox Biotech, a company that possess fundamental patents, granted in the US and in Europe and pending in Japan, which cover the rights to develop anti-diabetes drugs aimed to inhibit NOX. Glucox Biotech also owns international (PCT) substance patent applications on its first and second compound generation.
Anvari E, Wikström P, Walum E, Welsh N. The novel NADPH oxidase 4 inhibitor GLX351322 counteracts glucose intolerance in high-fat diet-treated C57BL/6 mice. Free Radic Res. 2015;49(11):1308-18.