Melanie Cole, MS (Host): A recent study from the Bass Lab has revealed how transcription factors within individual cells influence the identity and function of insulin-producing beta cells in the pancreas. This, according to findings published in Cell Metabolism.

Welcome to Better Edge, a Northwestern Medicine podcast for physicians. I'm Melanie Cole. We have a panel for you today with Dr. Joseph Bass, he's the Charles F. Kettering Professor of Medicine in the Department of Medicine at Feinberg School of Medicine, and he's the Chief of the Division of Endocrinology, Metabolism, and Molecular Medicine at Northwestern Medicine; and Benjamin Weidemann, he's a student in the Medical Scientist Training Program, which is a dual degree MD-PhD program at Northwestern University and he's the lead author of the study.

Thank you both for being with us today. And Dr. Bass, can you start by explaining the role of transcription factors in regulating gene expression, particularly in insulin-producing beta cells?

Joseph Bass, MD: Thank you for your interest. Transcription factors are one of the basic regulators within our body and in our cells of gene expression, which means they are responsible for taking the messages that are encoded in our DNA and turning those genes into functional entities, whether they be hormones, proteins or factors that determine the shape of our eye, heart, lung, other tissues.

Transcription factors are important in each organ system throughout life and conduct different aspects in our daily physiology. They are involved both in the development or organogenesis of tissues. They are involved in the function of cognition and all brain activity. And they're especially important in organs of the endocrine system, which produce hormones.

The transcription factors are the ultimate flexible response point, which determines how our body produces hormones at different times of day in response to what we eat, in response to what we drink, in terms of the salt content of our diet, and requirements for factors such as our daily blood pressure and the regulation of reproductive hormones. All of these processes are controlled by transcription factors. And one of the areas that has been best studied and best characterized is the function of transcription factors in the production of insulin, which turns out to be sort of the paradigm of how these factors work. And many of the basic principles about transcription factor activity were discovered through studies of the pancreas and diabetes.

Melanie Cole, MS: Well then, what motivated your research into transcription factors and beta cells?

Joseph Bass, MD: So, our research began with work in our laboratory nearly 20 years ago at Northwestern, focusing on an understanding, believe it or not, of how our sleep wake cycle, which is encoded by our body clock, controls both our weight and our metabolism. And the original genes that regulate our body clock were discovered in part at Northwestern. And one pathway through which those genes operate is the regulation of transcription. So in essence, it turns out that the factors that tell time each day in our body, and last night or two nights ago-- we had daylight savings time, so we are very aware today when our alarm clocks went off, it feels like it's an hour earlier. Well, the reason it feels as if it's an hour earlier has everything to do with transcription factors and how they work in the control of functions in our brain important in sleep and wake. But also, it turns out they are important in our whole-body metabolism, particularly in glucose metabolism.

So, the work really began when I moved to Northwestern from the University of Chicago and began to collaborate with colleagues here, Fred Turek, and especially Joe Takahashi, who had discovered the first genes for the clock and had produced and generated in that work mutations in animals in the core transcription factors that control the clock. And they approached me with the question of why it was that these animals, or what might be going on with these animals with respect to other body functions. And shortly after I became engaged in that work, I recognize that one of the principal abnormalities in animals discovered because of their abnormal sleep-wake cycle was that they also developed unique type of diabetes in which the pancreas fails to produce insulin. So here, I went from an interest in understanding genes that are important in behavior to the link between that phenomenon and an endocrine disease, which is where my training had been, specifically pertinent to understanding how insulin is made in the body.

Melanie Cole, MS: That's fascinating, Dr. Bass. Thank you for telling us about the clocks that control gene transcription and how that dictates physiologic outcomes. So Ben, what did your single-cell sequencing analysis reveal about beta cell behavior?

Benjamin Weidemann: One thing I would add to what Joe was talking about in regard to how transcription factors operate is that they establish cell identity, but they also play a role in integrating these signals, nutrient status, hormone sensing, and stress, into gene expression. And this facilitates a dynamic response to the environment. But one way in which it does this is by interacting with other protein complexes, other transcription factors and co-regulators that alter DNA expression epigenetically through controlling the accessibility of DNA. And these interactions between transcription factors and their co-regulators is critical for maintaining health and disruption can lead to disease, as he sort of mentioned. But this brings us to, really, how we looked at how the cells were being activated, both at the single-cell level and across the 24-hour time cycle using circadian manipulations.

The single-cell sequencing, really, we used it because all these studies that Joe mentioned had classically been done in bulk tissue, where there's millions of cells of different cell types, some even play counterregulatory roles in metabolism. Some are acting directly inhibiting the other cell types. So, what we wanted to do was look at each individual cell and see what is the activity of these transcription factors that define the cell type and how does this associate with some circadian factors that we had some knowledge of. And what it allowed us to do is see that there are different subpopulations of beta cells that have different activities of PDX1. And this is a transcription factor that essentially tells a beta cell to be a beta cell and tells it to actually release insulin and synthesize insulin. So, what we found is that there's these high levels of PDX1 activity in some cells, and this corresponded with a low activity NF-kappa beta, a signal-dependent transcription factor that's associated classically with inflammation. These cell-to-cell differences really provide a window into understanding not only how these cells may interact with each other, how the programs within the cells interact with each other, but also give us the basis for understanding how inhibiting inflammation may be beneficial in certain cell types to promote insulin release.

So overall, really providing an insight into both cellular physiology and possible therapeutic approaches and really form the basis for future studies where we plan to look at how cell type-specific response to diabetes therapies may be modulated by the clock.

Joseph Bass, MD: I might want to just elaborate on Ben as his mentor in research. Ben, you have to understand, has a very sophisticated talent for learning and operating very complex computer programs and web-based information way beyond my own ability, which is more focused on understanding what the questions are within the context of disease.

So, just to fill in a little bit, if nothing else, what the story really that I started to tell about recognizing that animals that have abnormalities in their body clock also develop diabetes, occurred in stages, and this was over a period of many years. Another one of our students, Biliana Marcheva, used a tool that was very new at the time in which we could selectively, in an individual tissue, remove a gene. And when she did that, our prediction was, if we remove this body clock gene only from the pancreas, the animals would develop very profound diabetes. And in fact, that turned out to be the case.

So when we developed a tool to go ahead and extract this transcription factor, eliminate it from the pancreas, we found two major things. One was that the cells were abnormal in terms of their development. And the other was that they were abnormal in terms of their function. So if we skipped ahead into a period of about five years ago, really, a colleague of ours arrived here from the Salk Institute, Graham Parrish, who had been involved in the development of new approaches with gene sequencing, where we could obtain a much more detailed level of the code within individual cells that determines how they work. So, this would be as if we had read for the first time something like the Mayan code. And our ability to peer inside the cell had been revolutionized after the point at which we had already done the experiments to prove that the transcription factor was vital for the pancreas.

So, we took this approach of great resolution for reading the code in cells, and what we came to discover was that these body clock factors were sitting throughout the genes in the pancreas that we recognized as also having been identified in studies of how the pancreas cell learns to be a pancreas in development. And one such factor was called PDX1. And so, here we find that this clock, the hands of the clock, are sitting and controlling this early developmental factor called PDX1. So, our idea was that there must be some communication between how our pancreas tells time each day and how the cell develops. And therefore, what are the requirements to maintain the identity of the cell? And there are mysteries that can be opened from such a view. And one of the mysteries is what are the codes that are overlapping that tell the cell what its identity should be, how to develop into a pancreas and, at the same time, tell the cell how to perform in response to the day-night cycle.

And when Ben started to do this, what he found was surprisingly that the secret codes where these two processes interact had something to do with inflammation. So, that means innately within this tissue are the master control nodes for determining whether the cell goes through a series of events that turn on or off genes that are essential for both survival and for inflammation.

Our thinking is still early about this observation or discovery. But one of the implications of this is that, since type 1 diabetes is a disorder in which there's sudden loss and triggering of inflammation in these cells, that perhaps this has something to do with these secret messages that are normally latent or hidden within the cell. And they are somehow hidden through this interaction between a developmental and a timekeeping set of interlocked transcription events factors. And that these factors, when they're properly functioning, are burying this inflammation signal below the surface. And somehow when this starts to splinter, number of things can happen. We can either lose the function of the cell or the cell may go through death and destruction.

And so, we think that while the formal language that we sort of describe our science has to do with timekeeping and how our watchmaker approach to biology has been an experience with technology and single-cell sequencing, as you mentioned. We've learned something maybe even much broader, which is that the fundamental signals that determine how cells survive and respond to attack have something to do with the interaction between the instructions for development and the instructions for timekeeping, two of the most ancient processes in photosensitive mammalian cells throughout evolution.

Melanie Cole, MS: Absolutely fascinating. The broader implications that you were just discussing, Dr. Bass. And how they might impact diabetes research further on down the line. I'd like you to take us from bench to bedside and put this right into the patient's hands. Where do you see this research affecting, especially type 1 diabetes as you said, because the timing is such a factor? Give us a little bit of those broader implications and how it's really going to affect patient care.

Joseph Bass, MD: My idea or my take on that question would be the more we learn about instructions for how a cell, such as a beta cell, can operate optimally, the more we may be able to recapitulate those crucial interactions in a dish. Now, we know that regenerative medicine or cell-based therapies are an emerging technology in the area of type 1 diabetes, but also pertinent to other forms of diabetes because even in type 2, the most common form of diabetes that's typically associated with weight gain and diet. The critical event that determines whether or not individuals go on to progress to diabetes has to do with the extent to which the beta cell maintains its functionality. So, we think that we're learning something, and there are implications in both arms of the diabetes spectrum.

And what we might learn in a bench-to-bedside way, most approximately, may have to do with cell-based therapy. Some of the approaches in cell-based therapy are paying a lot of attention to the ideas that are emerging in studies of the clock system in the beta cell because these factors can be chemically and genetically manipulated safely to enhance the stability of cells that produce insulin and to promote the health in the theory of replaced beta cells.

Benjamin Weidemann: And Joe, I don't have any corrections. But I'll just also add that, in addition to regeneration, there's always a potential for, as Joe mentioned, towards the end of type 2 diabetes or the one typically caused or associated with obesity, which occurs later in life, we still don't know exactly how these cells fail. Others have looked at similar approaches as what we've used here and see that there is some alteration in the cell populations and end-stage type 2 diabetes. We suspect that some of the programs we identified in our study are important in that process. Therefore, especially for those who have had diabetes for a long time, some of the pathways we identified may be intervened upon to help promote the survival of their beta cells, perhaps even the function of their beta cells, which may alleviate some of the burden of having always to take insulin or some of the negative side effects other type 2 diabetes medications.

Melanie Cole, MS: I'd love to give you each a chance for a final thought. So Ben, starting with you, future directions for this research as Dr. Bass spoke about bench to bedside, where do you see the research going?

Benjamin Weidemann: Yeah. I think that we have just started to identify how cells are different from each other within what we typically thought of as a single population. So, there are multiple types of beta cells. We're still in the early stages of understanding what fully underlies these, certainly PDX1 and inflammatory signaling is part of it, but how PDX1 and inflammation vary across the day and how external signals such as other hormones or nutrient signals influence us throughout the day, how those feed upon these cycles and these transcription factors will be important to understand in the future. And I think doing this at a single-cell level will provide us with unique information that we typically haven't been able to obtain before the advancement of these technologies.

Joseph Bass, MD: I think we should also add Ben is embarking on the next phase of training. So, he's the future. It's also an opportunity to learn about basic disease principles. So hopefully, in the future, the experiences had studying this system will re-emerge and help him approach problems both as a clinician and as a scientist in the future.

For my own sort of group and interests, I think what we're keen to learn about more is the possibility that manipulating this system or studying its effects on memory of cell identity and survival can be approached in the context of human regenerative experiments. Now, it's important also to add that a unique aspect of the studies in the beta cell is our ability to move back and forth between experimental models in genetically defined conditions in rodents and small animals.

In addition to an opportunity, we've been grateful to have to participate in the national network in which cadaveric donors from individuals who have unfortunately died, have provided their tissue to both therapeutic and investigative efforts nationally. So, we are part of that consortium. As a result, what we learn may be closer to telling us something about how the systems regulating gene expression and orchestrated by transcriptional pathways are mediating functions of human tissues, and that's unusual. There aren't so many conditions in which this type of opportunity exists. Many more than there used to be, but it's still a unique window into human biology and an important disease process that is diabetes.

Melanie Cole, MS: Well, thank you both for joining us. And I hope that you'll join us again and update us as this fascinating research continues. To refer your patient or for more information, please visit our website at breakthroughsforphysicians.nm.org/endocrinology to get connected with one of our providers.

That wraps up this version of Better Edge, a Northwestern Medicine podcast for physicians. I'm Melanie Cole.