Wisconsin Public Television University Place  Michelle Kimple Targeting the Beta Cell in Diabetes Therapeutics  September 12, 2012 UP766  cc >> Good evening, everyone. Welcome to Wednesday Nite at the Lab. I'm Cassie Immel from the Biotechnology Center. This evening we have Michelle Kimple. She has received her PhD as an assistant professor of medicine in the Division of Endocrinology, Diabetes and Metabolism at the University of Wisconsin School of Medicine and Public Health. She received her bachelor's degree from Michigan State University and her PhD from the University of North Carolina at Chapel Hill. Before coming to Madison, Dr. Kimple completed her postdoctoral training at Duke University in the Department of Pharmacology and Cancer Biology where she transitioned her work on the G protein signaling cascades into cell and mouse models of insulin secretion and diabetes. So, without further ado, here's Dr. Kimple. [APPLAUSE]  >> Hi. Can everyone hear me? >> Yep. 

>> I'd like to, even though Tom Zinnen's not here, I'd like to thank him for inviting me to come give this presentation, and I'm really excited to tell you a little bit more about our work that we do. I just started my faculty position here in February. So a lot of what I tell you today is going to be background in some of the new and exciting areas that we're hoping to get into. And, as part of my training, I think that it's really important to give students the opportunity to talk a little bit about what they're doing. So one of my undergraduate research assistants is going to give the first 10 minutes of this talk. Her name is Allison Brill, and she's currently applying to medical school. So I thought that this would be really useful for her as the first part of the talk is going to be more about normal physiology and the pathophysiology of diabetes. So these are going to be things that she's going to need to know in med school. [LAUGHTER] So, here she is. And just, while she's coming up here, I just wanted to point out that this is a really cool picture of a pancreatic islet. 

And the islets are the groups of cells that contain the beta cells, and in this picture, the beta cells are red. And this is a human islet because the glucagon producing alpha cells, which are stained in blue, are distributed throughout the islet instead of just around the outside. And then the delta cells, which we aren't really going to talk about today, those are green, and those produce and secrete somatostatin. So, we're going to talk about beta cells and islets, so I thought it would be nice to explain what the picture was.  >> All right, can everyone hear me okay? Good. Well, as Dr. Kimple just mentioned, my name is Allison Brill, and I'll be giving the first portion of today's talk. So, we'll start out today by looking at a few learning objectives that we have. And we'll be beginning with understanding the pancreatic beta cell in insulin secretion as well as its role in the maintenance in normal blood glucose levels. We'll then move on to understanding the beta cell's role in type 2 diabetes and talk about some current type 2 diabetes therapeutics available that target the beta cell specifically. We'll gain some familiarity with GPCR, or guanine nucleotide binding protein receptors, and finally gain some insight into potential new therapeutic targets for type 2 diabetes. 

So, to begin, of course, we're curious why is it important that we find new therapeutics for type 2 diabetes, and why is this something that we should be spending our money on researching? Well, we have here a figure created in 2009 by the CDC, and this figure is outlining the percentage of adults in each county across the United States that have been diagnosed with type 2 diabetes. And, overall looking at this map, it's clear that type 2 diabetes is extremely prevalent all across the country. And, in fact, in some counties even greater than 11% of adults are type 2 diabetic. Now, looking a little closer to home, here we are in Dane County, Wisconsin, and we're somewhat a little island of health being in the zero to 6.5% range of type 2 diabetic adults. And, of course, this is good for us, but, unfortunately, we're not in the majority. And, in fact, studies have shown that in some demographics now, the percentage of children and adolescents with type 2 diabetes even exceeds those with type 1 diabetes. So, obviously, the availability of new type 2 diabetes therapeutics has the potential to help many people. So, as you probably all are aware or as is well known, type 2 diabetes has been linked with obesity. And we can look here at the trends of the distribution of type 2 diabetes across the country. And now we'll look at the trends of, excuse me, obesity across the country and see how the two compare. 

So, we're looking here, as I said, at obesity trends among US adults across the country, and this is over a span of 24 years from 1985 to 2009. And, as you can see, each year, more and more states are becoming more obese. And, in fact, in the last five years shown in this progression, Wisconsin alone moves from the 20% range to the 25% to 29% range of obese adults. And so obviously this is a pretty significant jump. And looking, again, at the trends of obesity across the United States, we're beginning to see something very similar that we saw in the type 2 diabetes trends. So, looking at these maps side by side, it's very interesting and, in fact, very telling to see how these two maps nearly perfectly overlay each other. And, of course, this is not to say that obesity is the sole cause of type 2 diabetes. And, in fact, it's still not perfectly understood why this obesity epidemic is occurring, but regardless of why it's occurring, it's clear here that obesity is definitely a factor in the increased prevalence of type 2 diabetes. So, to understand better what's happening in type 2 diabetic individuals and how this is related to obesity, we first have to understand what is occurring in healthy individuals in relation to their hormonal regulation of blood glucose levels. So, after we eat, the carbohydrates in our food are broken down into glucose. 

And this causes a spike in our blood glucose levels. And this spike in our blood glucose levels signals to the pancreas and specifically the pancreatic beta cells to secrete insulin. Insulin is a hormone. You've probably all heard of it. It has a couple different targets within the body, one of which being the tissue cells. So insulin signals to muscle cells and fat cells for the uptake of glucose. Insulin also signals to the liver to convert glucose into its energy storage form of glycogen, and insulin also acts back on the pancreatic alpha cells to inhibit glucagon production. And glucagon is a peptide hormone, usually secreted in conditions of hypoglycemia, or low blood sugar. And glucagon has the opposite effects of insulin. So, normally then, after the secretion of insulin, blood glucose levels are lowered, and this signals to the pancreas to stop insulin secretion. 

In obese individuals, there is an increased need for nutrients to maintain their body weight. And this increased need for nutrients causes an increase in blood glucose levels, and this increase in blood glucose levels then causes an increase in insulin secretion. And after time, this prolonged increased insulin secretion can cause something called insulin resistance in the body. And this is what is known as a prediabetic state. So, essentially, what insulin resistance is, is that the tissues, or in this case the liver, muscle, fat, and alpha cells do not respond to insulin as they would normally. So, in these insulin resistant individuals, they do have the ability to maintain their blood glucose levels within a healthy range as long as they are able to secrete more insulin to compensate for this insulin resistance in their tissues. And this is shown here diagrammatically. So, we have on the left, healthy individuals. Their blood glucose and insulin levels are normal, healthy. Their beta cell mass and beta cell function are also normal. 

And the beta cell function being, of course, the ability to secrete insulin. Moving to the insulin resistant or prediabetic state, we see that the blood glucose levels are still within a healthy range. However, we do see that the plasma insulin levels have increased. And this is due to the beta cells themselves being able to increase their mass or multiply within the pancreatic islet. So, they becomes a greater number of beta cells in the islet. And these beta cells, as you can see, still maintain their normal function. So more beta cells secreting insulin causes this increase in plasma insulin levels. In a subset of individuals, there becomes a point in which the beta cells are no longer able to compensate for the insulin resistance in individuals that are prediabetic, and this is characterized by the loss of beta cell mass as well as the loss of beta cell function. So, the beta cells undergo cell death, and there become less beta cells within the pancreatic islet, and the beta cells that there are are no longer able to effectively secrete insulin. And this accounts for this drop in plasma insulin levels here. 

And, as a result, the blood glucose levels increase. Interestingly, there do exist a subset of individuals who are able to maintain this insulin resistant state throughout the entirety of their lives, and a thought of why some individuals progress to type 2 diabetes while others stay in this insulin resistant state is that there is an underlying beta cell defect in the individuals that progress to type 2 diabetes, and this predisposes them to becoming diabetic. So, I would also like to point out that there do exist type 2 diabetes therapeutics that do not target the beta cell specifically. And one of which is insulin injections to compensate for the insulin resistance. Another of which is a drug called metformin, and this is a tissue sensitizer. So it sensitizes the body tissues for uptake of glucose, and this is usually used in conjunction with other type 2 diabetes therapeutics. However, today we'll be focusing on those drugs that specifically target the beta cell as that is what we work on in lab. So, now that we understand somewhat more of the role of the beta cells and the pathogenesis of type 2 diabetes, we can look more specifically at how glucose stimulates these beta cells to secrete insulin. So we have here a generalized schematic of the exterior of the beta cell. And under normal conditions, these potassium ions here are able to flow out of the beta cell through this ATP sensitive potassium channel. 

When blood glucose levels are raised, glucose moves through the glucose transporter and into the beta cell. And the glucose inside of the beta cell is then metabolized into ATP, and ATP is an energy source for the cell. So this increased concentration of ATP within the beta cell acts specifically on this potassium channel. And this inactivates this potassium channel, and so the potassium ions are no longer allowed to move out of the membrane and out of the beta cell. When inactivating this channel, it causes a depolarization of the cell. And this depolarization is what then stimulates another channel on the membrane, the voltage gated calcium channel here. And this allows this channel to open and allows calcium ions to flow through into the beta cell. So, this increased concentration of calcium ions along with the increased concentration of ATP together are what allow beta cells to exocytose or allow secretion of insulin out of the beta cell. And the first drugs to be targeted at the beta cells were these drugs called sulfonylureas and meglitinides, and these drugs act on this potassium channel. And what they do is they cause a prolonged activation of this channel which causes a prolonged depolarization of the cell, which I mentioned is necessary for the secretion of insulin. 

So, this causes a higher level of insulin to be secreted from the cell. Now, the problem with these drugs are that they are glucose independent, meaning no matter if your blood glucose is high or not, these drugs will cause your beta cells to be secreting insulin. And this has the potential to cause hypoglycemia or low blood sugar levels in individuals. So, the search for new type 2 diabetes therapeutics continues, and an interesting finding actually occurred during a glucose tolerance test given to both type 2 diabetics as well as non-diabetic individuals. And these graphs are depicting the amount of insulin secreted over time. And we see here that they were given glucose both orally as well as intravenously, and the IV administered glucose would bypass the gut and go straight into the circulation. So it's interesting, we see in the non-diabetic individuals that there's a large significant increase in the amount of insulin secreted when glucose was given orally as compared to when given through an IV. And the amount of glucose given, as you can see, is 75 grams. And just for reference, this is standard for a glucose challenge, if any of you have ever taken a glucose challenge, and this is about two times the amount of sugar that's in a can of Coke. So, we see this increase in insulin secretion when the glucose is given orally in the non-diabetic individuals. 

However, when we look at the type 2 diabetic individuals, not only has the stimulation of insulin secretion decreased, it is also delayed. And so we know that there's something in these type 2 diabetic individuals, a defect in the beta cells that not only is a defect for insulin secretion overall but is a defect in insulin secretion when glucose is administered orally. So we know that there's something occurring in the gut that is causing a stimulation of insulin secretion. And this led to what is known as the incretin hypothesis. And incretins are molecules that are secreted from the digestive tract in response to glucose that act on the pancreas. And the important part here is that these are secreted in response to glucose, and this is something that the drugs I mentioned earlier, the meglitinides and the sulfonylureas, were not able to do. So, now Dr. Kimple will talk to you more about the overall scheme of the incretins as well as how they are related to the research that we do in lab. [APPLAUSE] >> Thank you, Ali. Well, that was, oh yes? >> What would cause the defect of the beta cell? >> There's several ideas. There's people who have defects in the potassium channel. 

That tends to cause something that's a more extreme form, an earlier onset form of diabetes. But a lot of work is going into some of the signaling pathways that I'm going to talk about later that are not necessarily the primary stimuli for insulin secretion but more modulate insulin secretion. So it could either be a defect in one of those primary components of the canonical insulin secretion pathway or it could be a defect in how these pathways are modulated. >> So you would inherit that defect? >> Um, yes. That's sort of the idea is that, and this isn't really my field of expertise but this is sort of the general idea now is that up until recently we haven't been challenged in this way with this amount of the prevalence of obesity in insulin resistance in the country and really worldwide. So there was really no selective pressure for these different genetic variants I wouldn't say that they're mutations. And it's certainly, for the most case, it's not just one. When they do these genome association studies, they're pulling out 20-30 genes that might have a 1.1-fold increased risk for diabetes, but when you add them all up, you're still increasing risk. But then there's a whole other camp that maybe this is something that's environmental too, environmental infectious. I think the lifestyle definitely contributes to obesity. 

Could lifestyle also be contributing to more transient beta cell dysfunction versus just genetics? You think about that as something that you're born with. You have it your whole life. Is there something that's impacting the DNA in a more transient manner? Yeah? >> When the obese person moves toward a normal life, are these effects mitigated? >> Yes. So, even people who are newly diagnosed type 2 diabetics have the ability to restore or to become non-diabetic once the insulin resistance is gone. So, in particular, I think some of the most dramatic results are with gastric bypass surgery, and that's not just because of weight loss. It actually happens within a week or so of the surgery itself. And some people think that some of these incretin pathways are playing a role. When you actually cut off the food moving to that proximal part of the intestine, you're ramping up incretin signaling. It's almost like, I don't know It's a little bit confusing if you think about it because it's stimulated by glucose, so you'd think that maybe you'd have decreased incretin signaling, but it actually seems that there might be more. And maybe it has something to do with more of the sugar actually making it to the colon, which is where these incretin molecules are being secreted. 

Yeah? >> So where would that leave the non-obese 20% who are diagnosed with type 2 diabetes. >> Well, that's a very good question and is probably why obesity certainly can't be the only explanation and why people are still talking about, I'm trying to think of some of the, like cytomegalovirus and other viruses that might be involved in increased risk. A lot of people think that the hygiene hypothesis might be playing a role in type 2 diabetes and in a lot of other conditions too. It's clearly and open book as to what exactly is happening. >> And what was that hypothesis? >> The hygiene hypothesis. Because everything we use now has antibiotics in it that we're not exposed to normal healthy levels of bacteria like we used to be 20-30 years ago, and that that's actually causing the increased food allergies, asthma, type 1 diabetes is increasing in prevalence independent of obesity. And type 2 diabetes also has an immune component too. I'm not really going to talk about it, but they're not as separate of diseases as they used to be. They might have different ideologies, but in the end it's still an immune mediated beta cell destruction. Even though type 2 diabetics generally don't lose as many of their beta cells as type 1 diabetics do. 

But if you lose enough of them, you're still going to need insulin injections at some point. All right, so, as I'm going on, if you feel like you need to ask a question, please feel free to interrupt me. But, of course, there will also be time at the end too. So, I think it's sort of interesting to go through a little bit of history sometimes. So I want to talk about the history of the incretin hypothesis. I didn't actually realize that this was around for as long as it was. It's been an idea for more than a hundred years. So, in 1902 a report by Bayliss and Starling sort of reported their hypothesis that gut derived signals after ingestion of nutrients elicit pancreatic endocrine responses that effect the disposal of carbohydrates. And, basically, that's exactly what incretins do. But it wasn't until the 1930s where another group Zunz and La Barre, prepared intestinal extracts and demonstrated that they could produce hypoglycemia in dogs. 

But other groups were unable to reproduce this effect in fasted animals, and so this report sort of died. They figured that it was some sort of an experimental artifact. And it wasn't until the 1960s, so another 30 years later, that there was the actual first biochemical demonstration of the incretin effect. And this sort of breakthrough occurred because of the development of precise assays for blood glucose in plasma insulin. I just have a little snapshot from this 1964 paper. And this looks very similar to the graphs that Ali showed except now we're looking at blood glucose as well as plasma insulin. And the intravenous glucose here is in black, and the intrajejunal glucose, which is being injected directly into the jejunum, part of the small intestine, is in the dotted lines. And you can see that the intrajejunal injection of glucose leads to a lower peak glucose and a faster resolution back to the baseline glucose levels. But if you now look at the plasma insulin, you can see why that occurred because plasma insulin secretion goes dramatically up, and in this paper they suggest that the incretin effect is actually responsible for about half of the insulin secretion capacity after an oral glucose challenge. So, these sort of seminal works led to the discovery of glucagon-like bioactive peptides, which is basically what incretins are, that are secreted upon feeding. 

About 10 years after the biochemical characterization Dupre and others purified the first incretin, which had been known previously as gastric inhibitory peptide, or GIP. And they show that when they co-injected glucose and GIP into individuals, they significantly increased their insulin secretion capacity as when they just injected them with glucose alone. And then later on in the 1970s, the glucose dependence of the GIP effect was demonstrated, and now it's been renamed glucose dependent insulin atrophic peptide. So they still use the same acronym. They just renamed it. And then in the 1980s there is a demonstration in several labs that neutralizing the GIP effect did not really fully ablate the incretin effect. So, there is the hypothesis that there were other bioactive peptides that might be more important than GIP, and, interestingly, GIP acts through a receptor on the beta cell, and GIP-based therapeutics have never been shown to work in type 2 diabetes. And this actually, a few years later, came to pass that with the discovery or the sequencing  of the human genome that the human preproglucagon gene family was sequenced, and this new incretin glucagon-like, peptide 1, was discovered. So, this is actually the same glucagon or proglucagon gene that expressed in the alpha cells, but in the alpha cells, the bioactive peptide is actually glucagon and it's specifically cleaved out of this longer propeptide sequence. In the alpha cells, or in the L and the K cells of the intestines, which express this prohormone convertase, they specifically cleave out this GLP1 molecule. 

So they're making two different peptides. Two different cells are making two different peptides from the same gene. And then, this is also a good place to point out that the full length bioactive peptide here is from residue seven to 36 of GLP1. And when GLP1 is cleaved by dipeptidyl peptidase 4, which happens fairly rapidly, probably to put a brake on that incretin effect, you lose these first two amino acids, and then the peptide doesn't function anymore. And that's just something to sort of, this was a good point to talk about it, but it's something to keep in mind for the next couple slides. So, in 1997 it was actually demonstrated that infusing glucagon-like peptide 1, or GLP1, can restore normal glucose control in type 2 diabetics. So here you're looking at blood glucose over time, over a 24-hour or 18-hour period. And I know most of you don't really know what the glucose really stands for, but this is in the stimulatory range for insulin secretion. Yet, these individuals aren't producing enough insulin in order to bring this down to baseline. So these are very elevated blood glucose levels. 

So this is in a diabetic that's been injected just with normal saline intravenous infusion. And then, here's a diabetic individual that has been infused with glucagon-like peptide 1 in that saline, and that quickly brings the blood glucose levels down. And they remain low ever after this person has eaten, which here, if you compare the size of these blood glucose spikes, they're dramatically lower in the GLP1 infused individual. So, this was the first indication that the incretins, in particular GLP1, could be potentially targeted for a type 2 diabetes therapeutic. So, just for the people who are more interested in more of the specifics of how the signaling pathway works, and now we're getting closer to what my laboratory actually studies, the receptor for GLP1 is the heterotrimeric G protein coupled receptor, or GPCR. So you can imagine, this is the cell membrane, the lipid bilayer, and GPCRs have seven transmembrane spanning domains. They sort of snake through the membrane. Of course, in a three-dimensional fashion. And the exterior of the C terminal end of the protein is exposed to the extracellular matrix, and the interior loops, or this is the N terminal end. The C terminal end extends into the cytoplasm. 

And they're called heterotrimeric G protein coupled receptors because they bind to heterotrimeric G proteins, and G proteins stands for guanine nucleotide binding protein. And guanine nucleotides, the most common ones are GDP, diphosphate, or triphosphate moiety. And the heterotrimeric comes from the fact that this protein is actually a complex of three independent proteins: an alpha, a beta, and a gamma subunit. The alpha subunit is the one that binds to the guanine nucleotide. And when that guanine nucleotide is GDP, it's inactive, it's bound to its regulatory subunits, beta and gamma, and it's also bound to the receptor. When an agonist binds, like GLP1 floating through the blood stream and making its way to the extracellular matrix of the beta cells, it binds to the N terminus and these extracellular loops and causes a conformational change in the protein that's transmitted to the G(alpha) subunit. The G(alpha) also changes its conformation, and it loses its affinity for GDP and releases it into the cytoplasm. It gains an affinity for GDP which is at 10-fold higher concentrations intracellularly. So this is just a purely stoichiometric process by where it binds to GTP. And once it's bound to GTP, it can release beta gamma, which have their own downstream signaling partners, but this G(alpha) GTP is now active to signal to its downstream effectors and to elicit biological changes. 

And once GTP is hydrolyzed by the G(alpha) subunit, because it's not only a signaling molecule, it's also an enzyme and it's enzymatic activity is to cleave this terminal phosphate and return it back to the GDP found state, this is when the signal returns back to baseline and it's ready for another round of activation. So, that's my G protein background, and of particular note is that the GLP1 receptor is coupled to GS and G proteins are classified based on their alpha subunit identity and there's only four classes of G proteins. So the ones that we're going to focus on, G(alpha)S is one of the two that we're going to focus on today. And the downstream effector of G(alpha)S is an enzyme called adenylate cyclase, and it converts ATP, the energy source for the cell, into cyclic AMP. And cyclic AMP, itself, can act on numerous downstream signaling pathways to elicit biological effects. And this is shown in the next slide, which hopefully can orient you a little bit more to what's happening in the beta cell. So you see the seven transmembrane G protein coupled receptor, the GLP1 molecule that's been bound to the extracellular side of the receptor which is subject to cleavage by this dipeptidyl peptidase 4 enzyme which will inactivate it, but while it's active and bound to the receptor, it signals to G(alpha)S to stimulate adenylate cyclase activity and lead to an increase to intracellular cyclic AMP levels. And cyclic AMP doesn't actually stimulate insulin secretion on its own. It requires ATP and calcium, which can only occur physiologically when glucose has been taken into the beta cell and metabolized. So, it really is a potentiator of glucose stimulated insulin secretion, and this is how the incretin effect is thought to happen in the beta cell. 

But what's interesting about cyclic AMP is that as opposed to the sulfonylureas and meglitinides, which basically just block the potassium ATP channel and force the beta cell to depolarize, cyclic AMP has other targets that might be involved in beta cell growth proliferation and survival. So it's possible that while it's still a little bit controversial, drugs that target these sort of signaling pathways might actually to be able to augment residual beta cell mass in type 2 diabetics or to preserve the beta cell mass that they have. And interestingly, there's two classes of therapeutics that have come out in the past decade that are actually used clinically, and so this sort of brings the whole story for GLP1 full circle from just the hypothesis that there is an incretin, what it does, to now type 2 diabetes patients are utilizing these drugs. So the one class of drugs are the GLP1 pneumatics, and probably the most well-known by its trade name is Byetta. Its generic name is Exenatide, and they're stable GLP molecules that are subject to cleavage by DPP4. Which leads me to the next class of therapeutics, the DPP4 inhibitors of which probably the first and most well known is Januvia or Sitagliptin. And now various pharmaceutical companies have one or both of these classes of molecules, and they're continuing to develop drugs that target the GLP1 receptor in sort of similar and, they like to think, better ways but probably the most different type 2 diabetes therapeutic that's come out recently, and this has all been since 2005, 2006. So something to note is that these are definitely very useful, clinically, but not all diabetes patients respond to Exenatide or Sitagliptin or related compounds. So, what I'm interested in are the G(alpha)I subclass of G proteins which now, if you look at what their effects are, they're to block adenylate cyclase and decrease cyclic AMP production. So, when I came to Wisconsin, or really even during my postdoc and since I've been here, my sort of thoughts are, is there an endogenous signaling pathway that acts through one of these other G proteins that's actually interfering or blocking with the action of these GLP1-based therapeutics because they act on the same downstream signaling pathway, just in opposite directions. 

And is it possible, if you could discover this G protein in the receptor that it's coupling to, could you target that either just in general or could you use it to augment the effects of these other drugs? So, where to start? There's so many G(alpha)I coupled receptors, and most of these receptors up here have been shown to have functions in the beta cell that led to decreased insulin secretion. So, the smaller problem is actually looking at the G protein itself. So, there's, at least in non-specialized cells like the eyes, the retina, and taste buds, there's a limited set of G(alpha)I subfamily proteins. And the one that I started working on in my postdoc is called G(alpha)Z, and really the most that had been known about it at the time was it was the only G protein that was resistant to inactivation by pertussis toxin, which actually puts this big ADP ribose moiety right on this cysteine residue here which G(alpha)Z, even though it's conserved in the other family members, G(alpha)Z doesn't have this cysteine. So this was part of the pathophysiology of pertussis, but it's also a useful biochemical tool for separating G(alpha)Z signaling from other G(alpha)I subfamily members. G(alpha)Z has other unique characteristics limited to tissue distribution, slow GTP binding rate, but interestingly, it has a very slow GTP hydrolysis rate. So once it becomes activated, its halftime for deactivation is about 10 minutes. And it had some potentially unique effector molecules described. But really little has been determined about its in vivo function. And the reason that I talk about pertussis toxin and how it's a useful biochemical tool is I was really interested in trying to find an endogenous or a native receptor in the beta cell that could specifically couple to G(alpha)Z and not any of those other G(alpha)I subfamily members. 

And I thought that I found one, and it turned out to be the case that PGE1, which is an endogenous E prostanoid molecule, and E prostanoids are sort of inflammatory related molecules that had been shown to endogenously block insulin secretion from the beta cells, that now we're looking at in vitro assay, so in a cell culture plate with beta cells, isolated beta cells, and seeing how well they can secrete insulin in response to glucose. So just glucose alone, you set that level at 100%. And PGE1 can block the amount of insulin that these beta cells can secrete. And then an unrelated compound also has this effect of being able to block insulin secretion. But when you preincubate the cells with pertussis toxin to inactivate all of the other G(alpha)I subfamily members, this other compound completely loses its effect on insulin secretion, but yet, the effect of the E prostanoid molecule, PGE1, remains. So this really suggested that the E prostanoid receptor in the beta cell, which at the time I didn't know what the identity of this receptor was, was specifically coupled to G(alpha)Z to block adenylate cyclase, the production of cyclic AMP, and downstream signaling pathway, and this other compound, which acts through the alpha 2 adonergic receptor, is coupled to a different G(alpha)I protein. So this was really my negative control because I knew that this receptor was coupled to a different G(alpha)I subfamily member. And now, looking at endogenous pancreatic islets. So, if you remember my beautiful fluorescent picture in the beginning, this is sort of the old school way of doing things where you actually use an enzyme. Instead of a fluorescent assay, you're using an antibody that's coupled to an enzyme that produces a colored product on these pancreas sections. 

And you can clearly see that the islet, this nice circle of tightly packed cells, is staining positively with an anti G(alpha)Z antibody, and that when you block the ability of this antibody to bind to G(alpha)Z, you lose this staining. So this, now we're looking at in vivo, like, fresh pancreas sections. This isn't just something that happens in cell culture. This is something that seems to be relevant to the pancreas, our primary pancreas cells in general. And this finding sort of led us to looking at mice that actually lack G(alpha)Z. So they don't produce, they're animals that have been designed to not produce the G(alpha)Z protein. And the idea is that if they're aren't producing this inhibitor of insulin secretion, that they should be able to secrete more insulin and clear blood glucose faster. So this is what we saw. The wild type mice, when you give them an injection of glucose, they have a peak, a blood glucose level that, sort of slowly over time, comes back down. And this is the similar pattern to what you observe in mice that don't express G(alpha)Z except that this peak is reduced, and the glucose returns back to normal slightly faster. 

And they actually have increased plasma insulin after glucose injection with really no difference at baseline, which sort of fits with something that's acting through this cyclic AMP pathway because, if you remember, cyclic AMP doesn't stimulate insulin secretion in the absence of glucose. And now, I don't know what happened. My PowerPoint crashed.  Sorry about this.  So, something that's nice about working with mice is you can actually isolate their pancreatic islets and perform these sorts of experiments in vitro. So in a test tube where you're sort of ignoring any potential effects of insulin tolerance in the peripheral tissues, and you're purely looking at secretion of insulin from the beta cells themselves. And, in fact, these islets that don't produce G(alpha)Z have an increased ability to secrete insulin in response to higher glucose concentrations than those from normal mice. And they actually show this increased cyclic AMP production that we predicted from this loss of an inhibitor of adenylate cyclase. So, I think at this point we confirm that G(alpha)Z was acting through an endogenous signaling pathway in the beta cell to block adenylate cyclase and block cyclic AMP production and its effect on insulin secretion, but we really had no evidence for whether this could be involved in the maintenance of beta cell mass. So we turned to a different model. 

As I mentioned earlier, or, actually, Ali mentioned this, that it's really the insulin resistance that causes the need for the beta cells to increase their mass to compensate for the insulin resistance. So, we thought that perhaps there was no difference in beta cell mass because all of the groups of mice that we'd studied up to this point were lean. So we subjected a subset of mice to a high fat diet study to induce obesity and insulin resistance. And then we did a lot preliminary studies before we started, but our idea was that these mice would be protected from developing diabetes. And that's what we saw after sort a long-term, for a mouse, high fat diet study, the wild type mice have become significantly more glucose intolerant than the control mice. So the wild types have normal G(alpha)Z expression, and, again, the G(alpha)Z knockout have no G(alpha)Z expression. And the high fat diet, wild type mice fed the high fat diet have a higher peak glucose and take longer to return to baseline. Even at three hours their blood glucose levels are still mildly elevated. Whereas, the G(alpha)Z-null mice that are fed the high fat diet really show no deficit in glucose tolerance. And the, I'm not going to show you this, but the effect on insulin secretion itself wasn't enough to explain the differences that we saw in these groups of mice. 

But what we did see is, if you remember some of those islet pictures earlier, the islets of both groups of mice seem to be significantly enlarged from the high fat diet. And here, the red is beta cells. So they're just sort of crammed in with beta cells and growing out into these elongated shapes instead of the nice circles that we saw before. But the G(alpha)Z-null islets from mice fed the high fat diet were significantly larger still, and they had these areas where we can actually use a marker for replication. So these are cells that are actively multiplying at this point in time. So this is sort of a snapshot of what's happening on that day. And even at the end of the study we were surprised we still saw so many replicated beta cells. If you quantify this, it seems that both the G(alpha)Z-null mutation plus the high fat diet feeding have this sort of synergistic effect on really increasing the replication rate and increasing the number of beta cells and the size of the pancreatic islets. And you could even see that visually here just by looking at, these are what my islets preps look like under the dissecting microscope. And just visually, the islets from G(alpha)Z-null mice fed the high fat diet seem significantly larger. 

So, I think that we are looking at this pathway as a potential target for not only augmenting glucose tolerance in insulin secretion in vivo, but also potentially as helping the agents that act through the GLP1 pathway to function better. And I think that I'm sort of going to cut, I sort of hinted at this earlier, but we think that this all has to do with this E prostanoid receptor and the PGE pathway. These E prostanoids that are related to inflammation, they're increased in a high fat diet model, they're increased in insulin resistance models, so you actually have increased production of the molecule that actually stimulates the beta cell receptor that's coupled to a G(alpha)I protein which we believe is specifically G(alpha)Z. So, because the expression of G proteins and the receptors varies between cells and the expression of G(alpha)Z is sort of limited in tissue distribution as compared to other G proteins, we feel like this might have some sort of increased potential for being able to make a targeted therapeutic for type 2 diabetes. And I see that it is already 8 o'clock, so I think I'm going to skip to the acknowledgments.  Actually, maybe I'll just talk a little bit about, so this is sort of continuing from the last slide where we're talking about this E prostanoid receptor G(alpha)Z coupled pathway that might be interfering with the proper functioning of this GLP1 receptor. And even without doing anymore biochemical characterization, we feel like this is an important avenue to look at in diabetes patients and in samples from human diabetes patients and not just mouse diabetes patients. So I actually have just received a pilot grant from the University of Wisconsin Institute for Clinical and Translational Research to collaborate with the director of the Islet Research Lab here at the UW. His name is Luis Fernandez, and he and I are going to collaborate for him to provide both non-diabetic and type 2 diabetic islets from organ donors. But something that you don't always receive with those samples, or hardly ever receive, is a matched plasma sample. 

So we'll be able to correlate the function of the islets with if and how much this signaling pathway might be dysfunctionally up-regulated in these type 2 diabetic individuals. And then the Wisconsin Partnership Program, I just put in a grant to start a trial looking at living, normal type 2 diabetic, and even type 1 diabetic individuals to see whether the levels of the molecule that binds to this receptor in the plasma will correlate with the response of the individuals to agents that act through this GLP1 receptor pathway. So we're definitely pursuing this, but my PhD is in biochemistry so I'm really interested in understanding the molecular mechanisms that are underlying this signaling pathway. And not only am I interested in it just from a purely scientific standpoint, but I think that it's important to know how these molecules are precisely working, and perhaps you could even target them better if you understood that. And, basically, all that I've said currently is that they're acting through cyclic AMP. I'm sure that it's true, but it's very superficial because cyclic AMP has numerous downstream effects, and these effects can be different based on where the cyclic AMP is being produced within the cell. It just doesn't increase every where in the cell. I think you can see this in this representation here that it's a microgradient of cyclic AMP within this membrane microdomain. So, cyclic AMP also has different effector molecules. At least two of them are protein kinases which mean that they put phosphates onto other molecules, and they cause them to either be activated or deactivated. 

So the identity of those proteins is going to be very important, and do they differ between the cyclic AMP levels that are increased with G(alpha)Z loss and the cyclic AMP levels that are correlated with this GLP1 receptor pathway. And another important downstream signaling pathway is transcriptional regulation. Just in a preliminary study with activated G(alpha)Z, it seems that the cyclic AMP response element modulator, which is a transcription factor affecting the transcription of numerous genes, might be related to part of the effect that we see in the G(alpha)Z knockout mice. So, we have some current directions looking at gene expression using these new affymetrix exon arrays. If you have ever heard of gene ships or gene arrays, these are very similar except that you're not only looking at overall gene expression but also at specific splice variance of genes because you could have difference in splice variance but not really be impacting the overall gene expression level. So we're excited to use some of these new technologies and to compare the effect of GLP1 treatment in islets from either G(alpha)Z-null mice or wild type mice. And then another thing that I already mentioned, but these proteins phosphorylation events are also something that's becoming easier to be able to sort of tease apart. So it's actually a technology that we're getting started with collaborators here and at Georgia Tech University. So, I think I will just skip to the end. So, the time is right for new therapeutics. 

There hasn't been a type 2 diabetes therapeutic, a new diabetes therapeutic in almost a decade. These red dots here are sort of questions about the therapeutics that are out on the market now. So, I think that the time is good start looking at some new and different pathways. And then, my acknowledgments. This is my little lab that I have here. I started my faculty position in February, and you heard Ali talk, and then Mark and -- are undergraduates. Renee and Nate are technicians, and Josh is my first grad student in the nutrition program. And since my space isn't ready yet, I've been hanging out in the Alan Attie lab. If any of you know about the research that's going on on campus in diabetes and obesity, he's one of the leaders in the field, and he's been very helpful and kind letting me squat in his space while I get things up and going. And then my clinical collaborators, Dawn Belt Davis, Melissa Meredith, and Luis Fernandez. 

And then my old colleague from UNC Chapel Hill who's now a mass spectrometrist who's interested in the phosphoprotein profiling of G proteins, luckily for me, is Matt Torres. And then I have had and currently have funding from many different sources, both for my basic research program as well as my clinical and translational research program. So, just as a plug for my husband, he's going to be talking soon, but he made us a Kimple labs website. If you want to learn more, you can go to KimpleLabs.com, and you can pull up a link to both my lab and his lab's website and what we're working on. So, I'll take any questions now. [APPLAUSE]