Intolerable secretion and diabetes in tolerant transgenic mice, revisited

A new mouse model linking diabetes, insulin secretion and autoimmunity with a high-fat diet supports a shared mechanism for type 1 (T1D) and type 2 (T2D) diabetes. In this model, the protein secretion system of insulin-producing pancreatic beta cells is stressed, leading to increased beta cell apoptosis and diabetes via reduced levels of the transcription factor GLIS3, a pathogenic pathway that can be mimicked by a high-fat diet.

Read more of this commentary in Nature Genetics

BioCentury Innovations

Our work was highlighted by BioCentury Innovations as a novel strategy to screen for anti-diabetic drugs:

Nature research highlight

Yes, diabetes is a genetic disease

With the publicity of our recent Nature Genetics paper, I have been discussing diabetes with many journalists. Almost without exception they seem surprised that I call diabetes a genetic disease - and they are not alone - even among the medical community it is standard to (incorrectly) call diabetes a lifestype disease.

The genetic basis for diabetes has been long established. It really is a shame that diabetes is so often called a "lifestyle disease". It isn't. 50% of the susceptibility to type 1 diabetes is genetic, and 70% of the susceptibility to type 2 diabetes is genetic.

Most of the remainder of the susceptibility is environmental. For example, certain types of dietary fat, such as palmitate, increase the fragility of beta cells. It is quite plausible that much of the increase in diabetes incidence in the past decades is due to changes in diet that make our beta cells more fragile. But even when we are talking about dietary factors, I think it is important to recognise that much of the effect is environmental rather than lifestyle. "Lifestyle" is easy to dismiss by blaming the patient for their own choices. Calling diabetes a lifestyle disease is one of the reasons that research and medical advances in diabetes are lagging behind, and the stigma contributes to the ill effects of diabetes (e.g., increased risk of depression, more antagonistic relationships between patients and clinicians). Recognising diet as an environmental factor takes away this stigma, is more accurate and allows us to tackle the problem using public health approaches. For example, some of the highest rates of diabetes are in the poorest neighbourhoods of America. In these areas, junk food is cheap and available everywhere, while good food is simply not practical - it is rare, expensive and takes more time to prepare than many poor families have. Likewise, the neighbourhoods may not provide the physical infrastructure that allows for a mobile lifestyle (parks for kids, urban design that promotes walking, etc). It is not a "lifestyle choice" to live in these diabetogenic environments, but it is a public policy choice to allow these environments to exist.

To really tackle the diabetes epidemic we need to recognise that the root cause is not in personal choices made by individuals. The root cause is in the social structure that we have created, in the urban design of cities, the changes in food culture, the demands placed on our time. We have made unhealthy lives the easiest to live. We can either ask individuals to make heroic efforts to overcome these obstacles to a healthy lifestyle, or we can use public policy to make our food environment and urban environment more healthy. Some of these policy changes would be immediate and easy (e.g., changing the tax structure to make good foods cheaper and junk food more expensive, or regulating the removal of the most toxic components of junk food). Other policy changes will take generations - even if we require urban planning to take into account healthy lifestyle promotion, the turnover in infrastructure is so slow that it will take a long time to occur. We certainly need major medical advances, which hopefully our study will aid, because the social changes needed will take decades to fully implement. But that is no reason not to start the public policy debate now, and even the small first steps will save the lives of millions (as well as saving billions from health care budgets).

Diabetes research in the news

Our new story on diabetes research, published in Nature Genetics, has made the news this week, with TV apperances on VRT and Kanaal Z. There are also articles in English, Dutch (here, here and here), French and Spanish media.

Nature Genetics interview

Nature Genetics has an interview with me on their blog: 

The discovery that NOD^k mice with the insHEL transgene develop diabetes is described as being serendipitous. What were your initial thoughts about this? 

At the time we first found that NOD^k.insHEL male mice developed diabetes I was actually working on immune defects in NOD mice, rather than beta cell defects. My first thoughts were that this was just another immune defect, with the immune system attacking the beta cells because they expressed the insHEL transgene. Since it fit our preconceived ideas we didn’t take too much notice, but just to be safe I set up a backcross to eradicate the adaptive immune system from the NOD^k.insHEL mice. It took a couple of years for the mice to breed and age, so I had almost forgotten about the finding when the first immune-deficient NOD^k.insHEL mice started to develop diabetes. At that point I was really startled – the cross should have eliminated diabetes if it was immune-mediated. I knew then that we were looking at some completely new biology – which took another 10 years to dissect! 

What advantages does your new mouse model bring to the field?

There are so many aspects to diabetes that it is often impossible to untangle the causes of disease. For example, one of the critical clinical developments in type 2 diabetes is the death of beta cells. It marks a shift from insulin-resistant diabetes (which is largely treatable), to insulin-deficient/insulin-resistant diabetes (for which there are no effective treatments). But why are the beta cells dying? From previously mouse models there were many reasonable hypotheses that were put forward – maybe it is the demand placed on the beta cell for extra insulin production, maybe it is a toxic effect of high blood glucose levels, maybe it is a side-effect of the high fat diet used to induce diabetes in the first place, or maybe it is immune-mediated. Our model has the advantage that it can strip away all of these interactions to observe the direct effects of forcing beta cells to produce too much protein – a process that results in beta cell failure. Looking forward, I see a major advantage in using this model to screen for drugs that stop the loss of beta cells in type 2 diabetes, which is really the key unmet medical need in diabetes treatment.

One of your interesting observations is the difference in diabetes incidence between the male and female mice, mediated by male sex hormones.  What parallels are there with humans and how might you use this model to explore this further?

So far we have only seen diabetes in male insHEL mice, despite challenging female insHEL mice with multiple strategies that promote diabetes in male mice (diet, autoimmunity, genetic background). At a cellular level it looks like male islets are just under more metabolic pressure than female islets, such that the insHEL stress is enough to make male mice diabetic, while female mice stay healthy. This could actually explain a lot about the epidemiology of type 1 diabetes in humans. Most autoimmune diseases have a strong female bias, while type 1 diabetes has a weak male bias. Our hypothesis is that maybe males have an intrinsic islet fragility (perhaps from supporting a larger body mass), while females have an intrinsic susceptibility to autoimmune disease. In epidemiological terms, these two effects may cancel each other out, leading to similar levels of type 1 diabetes in males and females, but at a clinical level it may mean that different individuals would respond better to different treatment strategies.   

You identified two loci linked with insHEL-induced diabetes in the NOD^k mice.  What were your expectations about what you would find? Where you excited when Xrcc4 and Glis3 were identified as candidate genes?

Geneticists have been trying to work out the basis of spontaneous diabetes in NOD mice since the strain was first published in 1980. It turns out to be a very complex problem – there are more than 20 loci that contribute to diabetes susceptibility, and each time a loci is analysed in detail it ends up being a cluster of weaker loci working together. Decades later and we are only sure about a handful of candidates genes – so I didn’t have high expectations that we would progress far when looking at the genetics of insHEL-triggered diabetes. It turns out, however, that we had several major advantages. First, the genetics ended up being much simpler, with linkage only observed on two chromosomes. Second, because we knew which cell type was important – the beta cell – we were not operating in the dark about candidates. After filtering for expression in beta cells we were left with only a handful of candidates. Seeing Xrcc4 and Glis3 on the final list was bliss – they both made perfect biological sense. GLIS3 is one of the very few genes linked to both type 1 and type 2 diabetes in humans, and here we had it on our shortlist for a model that contains aspects of both diseases! It had taken more than 10 years to get to those two genes, but then we reached one of those dream runs in the laboratory where all the data just comes together, and every experiment gave support to the candidates.

You identify beta cell failure as a common link between T1D and T2D.  Are there ways that your findings can impact the clinical understanding or management of these diseases? 

In some ways, what we have here is the laboratory catching up to the clinic. The clinical overlaps between type 1 and type 2 diabetes have been apparent from the start, yet the research on genetics and animal models has consistently emphasized the differences. We may be in the process of reconciling these two approaches. The model that I favour is one where beta cell robustness or fragility lies at the centre of both diseases. In type 1 diabetes, failures in immune tolerance promote an attack on the beta cell, while in type 2 diabetes, hepatic insulin resistance leads to beta cell stress. In both cases, however, it may be the intrinsic robustness or fragility of the beta cell that dictate whether the pressure on beta cells remains subclinical or leads to diabetes. If this model holds true in patients then it would present a golden opportunity for preventing diabetes by increasing the robustness of beta cells.

New study may lead to improved treatment of type 2 diabetes

Genetic cause found for loss of beta cells during diabetes development

Worldwide, 400 million people live with diabetes, with rapid increases projected. Patients with diabetes mostly fall into one of two categories, type 1 diabetics, triggered by autoimmunity at a young age, and type 2 diabetics, caused by metabolic dysfunction of the liver. Despite being labeled a “lifestyle disease”, diabetes has a strong genetic basis. New research under the direction of Adrian Liston (VIB/KU Leuven) has discovered that a common genetic defect in beta cells may underlie both forms of diabetes. This research was published in the international scientific journal Nature Genetics.

Adrian Liston (VIB/University of Leuven): “Our research finds that genetics is critical for the survival of beta cells in the pancreas – the cells that make insulin. Thanks to our genetic make-up, some of us have beta cells that are tough and robust, while others have beta cells that are fragile and can’t handle stress. It is these people who develop diabetes, either type 1 or type 2, while others with tougher beta cells will remain healthy even in if they suffer from autoimmunity or metabolic dysfunction of the liver.”

Different pathways to diabetes development

Diabetes is a hidden killer. One out of every 11 adults is suffering from the disease, yet half of them have not even been diagnosed. Diabetes is caused by the inability of the body to lower blood glucose, a process normally driven by insulin. In patients with type 1 diabetes (T1D), this is caused by the immune system killing off the beta cells that produce insulin. In patients with type 2 diabetes (T2D), a metabolic dysfunction prevents insulin from working on the liver. In both cases, left untreated, the extra glucose in the blood can cause blindness, cardiovascular disease, diabetic nephropathy, diabetic neuropathy and death.

In this study, an international team of researchers investigated how genetic variation controls the development of diabetes. While most previous work has focused on the effect of genetics in altering the immune system (in T1D) and metabolic dysfunction of the liver (in T2D), this research found that genetics also affected the beta cells that produce insulin. Mice with fragile beta cells that were poor at repairing DNA damage would rapidly develop diabetes when those beta cells were challenged by cellular stress. Other mice, with robust beta cells that were good at repairing DNA damage, were able to stay non-diabetic for life, even when those islets were placed under severe cellular stress. The same pathways for beta cell survival and DNA damage repair were also found to be altered in diabetic patient samples, indicating that a genetic predisposition for fragile beta cells may underlie who develops diabetes.  

Adrian Liston (VIB/University of Leuven): “While genetics are really the most important factor for developing diabetes, our food environment can also play a deciding role. Even mice with genetically superior beta cells ended up as diabetic when we increased the fat in their diet.”

A new model for testing type 2 diabetes treatments

Current treatments for T2D rely on improving the metabolic response of the liver to insulin. These antidiabetic drugs, in conjunction with lifestyle interventions, can control the early stages of T2D by allowing insulin to function on the liver again. However during the late stages of T2D, the death of beta cells means that there is no longer any insulin being produced in the pancreas. At this stage, antidiabetic drugs and lifestyle interventions have poor efficacy, and medical complications arise.

Dr Lydia Makaroff (International Diabetes Federation): “The health cost for diabetes currently exceeds US$600 billion, 12% of the global health budget, and will only increase as diabetes becomes more common. Much of this health care burden is caused by late-stage type 2 diabetes, where we do not have effective treatments, so we desperately need new research into novel therapeutic approaches. This discovery dramatically improves our understanding of type 2 diabetes, which will enable the design of better strategies and medications for diabetes in the future”.

Adrian Liston (VIB/University of Leuven): “The big problem in developing drugs for late-stage T2D is that, until now, there has not been an animal model for the beta cell death stage. Previously, animal models were all based on the early stage of metabolic dysfunction in the liver, which has allowed the development of good drugs for treating early-stage T2D. This new mouse model will allow us, for the first time, to test new antidiabetic drugs that focus on preserving beta cells. There are many promising drugs under development at life sciences companies that have just been waiting for a usable animal model. Who knows, there may even be useful compounds hidden away in alternative or traditional medicines that could be found through a good testing program. If a drug is found that stops late-stage diabetes, it would really be a major medical breakthrough!”

Read more: Dooley*, Tian*, Schonefeldt*, Delghingaro-Augusto*, Garcia-Perez, Pasciuto, Di Marino, Carr,Oskolkov, Lyssenko, Franckaert, Lagou, Overbergh, Vandenbussche, Allemeersch, Chabot-Roy, Dahlstrom, Laybutt, Petrovsky, Socha, Gevaert, Jetten, Lambrechts, Linterman, Goodnow, Nolan, Lesage, Schlenner**, Liston**. 'Genetic predisposition for beta cell fragility underlies type 1 and type 2 diabetes.' Nat Genet. 2016

New diabetes drug-screening model available

Aire creates immune tolerance via an unconventional transcriptional mechanism

by Dina Danso-Abeam

The white blood cells of our immune system defend us from infection, a function which is coordinated by T cells. Immature T cells are formed with an ability to attack random targets (an adaptation to the rapid evolution of microbes), which means that by chance some targets are "self-targets" (normal proteins part of a healthy body). As a consequence, these "self-reactive" T cells can atatck the body, so it is critical to prevent them from causing autoimmune disease. To prevent autoimmunity, the immature T cells are screened in an organ called the thymus (located just above the heart), in order to ensure that all self-reactive T cells are eliminated.

The Aire gene plays an important part in eliminating self-raective T cells, by expressing genes that are normally restricted to specific organs (eg, insulin in the pancreas) in the thymus, providing full coverage for screening against self-reactivity. In patients that have mutations in the gene Aire, the thymus cannot provide full coverage of self-targets and fatal autoimmune disease develops. One of the mysteries of how Aire functions is its ability to express thousands of genes like insulin in the thymus. 

It has previously been shown that Aire is a transcription factor (meaning, it can bind DNA to activate genes and cause the expression of proteins) which can activate the expression of other transcription factors. We hypothesized that these secondary transcription factors might mediate the expression of the thousands of Aire-dependent genes like insulin; in effect, we predicted that Aire creates a cascade of transcription factors that results in the expression of thousands of genes.

In order to test this hypothesis, we investigated whether such a cascade regulates the transcription and tolerance of pancreas-specific antigens (e.g. insulin and glucagon) in the thymus. In the pancreas, Pdx1 is the key transcription factor which drives the expression of insulin. Interestingly both Pdx1 and insulin have been shown to be Aire-dependent in the thymus, so it was possible that Pdx1 was acting as a secondary transcription factor in the cascade by which Aire expresses insulin. Therefore we generated mice that specifically lack Pdx1 in the thymus.

By generating these mice, we found that expression of pancreatic-specific antigens such as insulin, needed Aire expression in the thymus, but did not need the transcription factor Pdx1. These results suggest that the broad tolerance that Aire creates in the thymus is not mediated by a conventional cascade of transcription factors, but rather relies on an unconventional transcriptional mechanism.  

This work will be published in a forthcoming issue of The European Journal of Immunology as:

Aire mediates thymic expression and tolerance of pancreatic antigens via an unconventional transcriptional mechanism

by Dina Danso-Abeam, Kim A Staats, Dean Franckaert, Ludo Van Den Bosch, Adrian Liston, Daniel H D Gray* and James Dooley*

Juvenile Diabetes Research Foundation

Good news in funding appears to come in pairs. The Juvenile Diabetes Research Foundation is supporting the Autoimmune Genetics Laboratory through a Career Development Award. This is a grant that I am particularly happy to receive, not just for the science that will come out of it, but because I have been a long-time admirer of the JDRF, who tirelessly raise money for research on type 1 diabetes. They are not only the leading sponsor of type 1 diabetes research (spending over $1.4 billion on research since 1970), but also take an active role in coordinating researchers and integrating patient into trials to ensure that the best results come from the money spent. As a PhD student with Chris Goodnow, I always joined in the Walk for the Cure fundraiser, and JDRF sponsored my conference travel to the International Immunology Congress in 2004.

Now the JDRF is supporting our research project on the contribution of non-hematopoietic defects to autoimmune diabetes:

The Non-obese diabetic (NOD) mouse is one of the best studied models of common autoimmune disease in humans, with the spontaneous development of autoimmune diabetes. Similar to the way multiple autoimmune diseases run in families of diabetic patients, the NOD mouse strain is also susceptible to multiple autoimmune diseases, with specific disease development depending on slight alterations in the environment and genetics. These results demonstrate the complexity of autoimmune genetics – in both human families and inbred mouse strains there appear to be a subset of genetic loci that skew the immune system towards dysfunction and an additional subset of genetic loci that result in this immune damage affecting a particular target organ. In the case of NOD mice and type 1 diabetic patients these additional genetic factors result in damage to the beta islets of the pancreas. While the previous emphasis on type 1 diabetes was strictly on the immune system, this model suggests the important role the pancreas may play in the disease process. If certain individuals harbour genetic loci that increase the vulnerability of pancreatic islets to immune-mediated damage, the combination of immune and pancreatic loci could provoke a pathology not caused by either set of genes alone.

Current approaches to genetic mapping in both mice and humans are confounded by the large number of small gene associations and are not able to discriminate between these functional subsets of genetic loci. However, we have developed an alternative strategy for functional genetic mapping. Instead of mapping diabetes as the sole end-point, with small genetic contributions by multiple genes, we map discrete functional processes of diabetes development. This has three key advantages. Firstly, as simpler sub-traits there are fewer genes contributing, each with larger effects, making mapping to particular genes more feasible. Secondly, by mapping a functional process within diabetes we start out with functional information for every gene association we find. Thirdly, by mapping a series of functional processes and then building up this genetic information into diabetes as an overall result we gain a more comprehensive view of diabetes, as a network of genetic and environmental influences that cause disease by influencing multiple systems and processes.

In this project we propose to use the functional genetic mapping approach to probe the role of the pancreatic beta islets in the development of diabetes in the NOD mice. We have developed a transgenic model of islet-specific cellular stress which demonstrates that NOD mice have a genetic predisposition of increased vulnerability of the pancreatic islets to dying and hence the development of diabetes. This is a unique model to analyse the genetic, cellular and biochemical pathways that can be altered in the pancreas of diabetes-susceptible individuals, shedding light on the role the beta islets play in the development of disease.