[MUSIC] The evidence we have now discussed suggests that pro-inflammatory cytokines are common targets in therapies aiming at reverting, or even preventing, beta-cell failure in type 1 and type 2 diabetes. But how can that be achieved, and what drugs do we have available on the market to do that? In fact, there are several ways now we can antagonize interleukine-1 action. First of all, we can use a principle where we block the interleukine-1 receptor. And of course a most natural way of doing that is to using the endogynously produced, but now recombinantly produced human interleukine-1 receptor antagonist, which as you'll remember competitively binds and thereby prevents activation of the interleukine-1 type 1 receptor. Industry has also produced antibodies against that receptor, thereby preventing binding of the agonists and the activation of the receptor. There are even antibodies against the accessory protein that by preventing the docking of this protein to the receptor ligand complex would prevent interleukin-1 signaling. The other fundamentally different principle is not blocking the receptor but by neutralizing interleukin-1 itself. And here, of course, the most obvious way is to produce antibodies against the main human form, interleukin one beta, that will bind and neutralize interleukin one in the circulation or in the tissue in. There are also combined antibodies against both R one alpha and beta. There are antibodies that are made in a special way, in fact by fusing the soluble type 1 receptor with an IgG portion of an antibody molecule, which is called the IL-1 trap. And it has a very high affinity to free circulating and tissue residing interleukine-1. And finally soluble type 2 receptor has been produced that will also serve as a binder and a neutralizer of interleukin-1. The question now is does interleukin-1 antagonism work in diabetes? Let us first look at the evidence from type 1 diabetes. Now recall that at the time of the occurrence of type 1 diabetes, there has been a long pre-diabetic period with a progressive autoimmune destruction of beta-cell mass, so that at the onset of the disease, it is estimated that between 30 and 50% of the pancreatic beta-cell mass is left. So it may not be surprising that starting interleukine-1 antagonism at this late stage of the disease does not seem to have any overall beneficial effects on beta-cell function. However, if you analyze subsets of those patients treated with our one receptor antagonists with recent onset type one diabetes. There is in fact a two and a half times higher beta-cell function in a particular subset of these patients. But these data need to be reproduced and repeated in several other specially designed clinical trials that will address this question. Is it possible that we could improve these results of targets in interleukine-1 by also targeting the adaptive immune system? Recent studies in an animal model called the non-obese diabetic mouse, or the NOD mouse, suggests that this is the case. In this figure, you see. Captain marker showing the cure of diabetes in several groups of mice. You can see that in placebo treated mice as the very bottom there's of course no effect on diabetes cure. If you employ IL-1Ra the blue curve. There's a very very slight and discrete cure of diabetes, but not very impressive. If you now employ an antibody that is targeting and inhibiting the function of a certain subset of T cells that are expressing the CD3 molecule on the surface, which is essentially all T cells. Then you can see that you have a very marked and significant protection and reversal of disease. But if you combine that with the red curve with interleukine-1 antagonism you get much more rapid reversal of disease and actually complete reversal that lasts beyond the treatment period. So the interpretation of this data is that perhaps we need higher doses of interleukin-1 to be able to affect diabetes in recent onset type 1 diabetic patients. Or if maybe that therapy has to be started much earlier that is, for example in risk individuals. Or perhaps in a model where we introduce islets in transplantation. But it is also possible that our IL-1 blockade may have to be combined with immunomodulation of the adaptive immune system. In other words, that we need combination therapies. The next question is then, does IL-1 antagonism work in type 2 diabetes? And as you can see from this figure, in fact this seems to be the case. In the top left panel, you can see the effect of giving 13 weeks of IL-1 receptor antagonist, to patients with long standing type 2 diabetes. And what the graph shows is a reduction in glycated hemoglobin after four weeks and 13 weeks of therapy. On the upper right hand panel, you can see the effect on black glucose, and you can see that even within one week of therapy, there's a significant reduction in plasma block glucose in these patients treated with R one antagonism. In the lower left panel you can see that if you stratify the patients based on their body mass index, and thereby on their distribution volume, those patients that have the highest exposure to the drug have, in fact, a reduction in hemoglobin A1C of 0.8%, which equals many other modern and adjunct therapies in type 2 diabetes. So, how is this then accomplished? What are the mechanisms of action in type 2 diabetic patients? In this figure you can see in the upper left panel, the effect of interleukin-1 recept antagonist administration for 13 weeks on an index of beta-cell stress. Which is the processing of proinsulin into mature insulin. And you can see that the ratio between proinsulin and insulin is significantly reduced by the therapy. And the other panels you can see, that not only is the relentless reduction in beta-cell function that occurs in patients that are left on the same treatment over time prevents it, but in fact the therapy induces an increase in beta-cell function that is significant both when you test beta-cell function was an oral glucose load or with an intravenous glucose load. Is there any effect on insulin resistance? No, in this study we studied that very carefully and we could not detect any effects of the drug on insulin sensitivity. So it appears that blocking out one action within the pancreatic eyelid auses beneficial effects on beta-cell function in longstanding type 2 diabetic patients. The burning question is of course now whether we can further improve anti-inflammatory treatments in type 1 and type 2 diabetes perhaps by even looking at alternatives to anti-inflammatory biologics. And in the remaining part of this module I'd like give to you two recent examples of how we have approached that problem. One of which is to try to reset beta-cell gene expression as a novel therapy against diabetes. The pancreatic beta-cell is in no way a passive bystander to its own destruction. It is very clear it participates very actively in it's own demise. And that the exposure to cellular and metabolic stress causes profound gene expression changes within the beta-cell. For example if you expose pancreatic beta-cells in vitro for cytokines such as interleukin-1 and or interferon gamma. There are upper both are defensive teams that try to restore beta-cell homeostasis but also of course upper regulation of detrimental pathways that cause the eventual destruction of the cell. In addition to that there's a whole array of gene changes that are related to changes in transcription factors that is related to differentiation of the pancreatic beta cell and also even to the communication of the beta-cell, or the exterior such as a surprising secretion of many kinds from the beta cells that would lead to the attraction of the immune cell. So it is this balance between defensive and detrimental gene expression profiles that eventually lead to the destruction of the cells. And how can we reset that in a constructive way so that the pancreatic beta-cell is prevented from undergoing apoptosis? It occurred to us that an interesting family of proteins that might be able to do this is inhibitors of the so-called lysine deacetylases. So what are these molecules? Lysine deacetylases are enzymes that are very important in gene and protein regulation through, first of all deacytelating histones. As you may remember, the histones are the backbones on which the DNA molecules are wound. And in order to get active transcription, you have to have a loosening of the tightly wound DNA around the histone backbone. So the role of Lysine deacytylases is to remove those spaces that allow the transcription operators access to the loosely bound DNA. These group of molecules are opposed by another family of molecules called lysine acetyltransase. That introduces these bases and then allow the unwinded chromatin and, thereby, active to be in transcription. So if we want to affect beta-cell to be transcription that is stimulated by inflammatory cytokines, can we then use inhibitors of lysine deactylases to affect that teen expression profile. Let us first look further into the biology of these interesting proteins. It is now clear that, not only do these regulate histone deactylation, but also more than 4,000 proteins. So in fact, these molecules have important function in relation to the regulation of virtually all proteins in the cell. Lysine deactylases come in two major families, the classical lysine deactylases and the so called sirtuins, of which there are seven. These group of molecules are, structurally, very different because the sirtuins are dependent on NAD and are thereby important energy sensors in the cell. Whereas the classical lysine deacetylases are not dependent on NAD but rather on zinc, a metal iron that is important for enzymatic function and that resides in a zinc binding pouch in all the classical lysine deacetylases. There are 11 different members of lysine deacetylases. Class I, which is considering HDAC 1, 2, 3, and 8. Class IIb which is HDAC 6 and 10. Class IIa which is HDAC 4, 5, 7, and 9. And then Class IV which is HDAC11. The sirtuins actually constitute Class III. Interestingly enough beta-cells express all the classical HDACs. And interestingly also many of these HDACs are regulated by inflammatory. Why are these inhibitors interesting in relation to diabetes? Because we actually already have drugs that can affect these enzymes, and they have been used for a long time in particular types of cancers, such as cutaneous T-cell lymphoma, and certain neurodegenerative diseases. And they are now in clinical trial for many inflammatory diseases. We tested those molecules on isolated eyelets exposed to cytokines and could show that the cytokine induced apoptosis was completely prevented by the addition of inhibitors of these molecules. We've also tested them in animal models. Non obese diabetic mouse I eluded to before. And have shown that short term therapy using these molecules before the answer to diabetes can completely prevent development of type 1 diabetes in these mice. The mechanism of action is both dampening of the immune system and also by affecting beta-cell responses to inflammatory cytokines. So, this is one avenue perhaps that can be pursued in clinical trials, where we now need to test whether HDAC inhibitors can actually be effective in type 1 diabetic patients. The second example of novel approaches to try to prevent inflammatory beta-cell destruction is to try to understand why beta-cells selectively destroyed by the inflammatory infiltrate. [INAUDIBLE] and also in type 2 diabetes where there are resident and cells. All the non beta-cells in the eyelet are essentially exposed to the same inflammatory environment as the beta-cells. So why are all the beta-cells destroyed? If we can understand that, maybe we can devise novel therapies. We have used a model of deep beta-cell differentiation to identify genes are important for this process. Because clearly, differentiating beta-cells acquires a sensitivity to these inflammatory molecules. And by microarray status, we identified one interesting candidate protein, a divalent metal transporter called DMT1, that is important in this process. DMT1 is a metal transporter but in the beta-cells it is mainly transporting iron. Iron is, as you probably know from biochemistry, a very important catalyst of many cellular functions that are both beneficial for the cell, but can also via the reaction cause overproduction of a reactive oxygen species that can cause cellular damage. And so the hypothesis behind this study was, that if we knocked out the DMT1 transporter in pancreatic beta-cells, could we then protect them from inflammatory damage? First of all, we examined the biology of this transporter. And it is indeed regulated by the proinflammatory sickle need pathways that I discussed with you previously, the pathway in particular. And we could show that do indeed up regulate not only the expression but also the activity of these transporters in pancreatic eyelets. Both from rodents and from human necro donor subjects. We then went on and asked the question, if we now use chemical chelators of iron, can we then prevent inflammatory mediated eyelid reactive oxygen species production and apoptosis. And this was indeed the case. The more molecular approach was to knock down the DMT transporter using small interfering RNAs. We were also able to show using three different siRNAs against DMT 1 that we could significantly reduce cytokine induced beta cell apoptosis. The critical question was now, could we reproduce that in an animal model? So we generated inducible beta cell specific DMT1 knockout mice. And we could show that, indeed, eyelids from these mice were protected against inflammatory attack. When we now generate a state of inflammation in the eyelids using multiple low-dose streptococcus, we could also show that this model of tupe 1 diabetes was prevented using the DMT 1 knockout model. And even more surprisingly, how high fat feeding induced type 2 diabetes could also be prevented in this particular knockout mouse. And both of these cases were related to an increase in beta cell function. So in conclusion from these facts cytokine binding to interleukin-1 receptors on the surface of the beta cell lead to the up regulation both of the expression and the activity of these transporters that lead to increased iron transport into the cells. That increased our iron will catalyze the generation of reactive octan species that harm both insulin secretion and can be a signal for apoptosis in the beta cell. And we showed that we can interfere in that process both by chelating iron but also by manipulating the activity of the DMT1 transporter. Again, these studies of course need to reproduced in clinical trials and such initiatives are under way. Just to highlight that there is evidence from patient studies that this might be true we recently conducted a large survey In the Copenhagen heart study population, more than 10,000 individuals followed for 15 years. And we could show that normal subjects in the background population that have slightly elevated iron saturation in the blood run a much higher risk for development of diabetes later on in life. And this was reproduced in an independent series from the Copenhagen general population study of 30,000 individuals from the herd of cohort. So I think these studies lend hope to novel pathways of therapy that perhaps can be used in combination with specific biologics to prevent inflammatory beta cell destruction and thereby hopefully to cure and prevent type I and type 2 diabetes. [SOUND]
