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Steve: This is Steve Freed with Diabetes In Control and we are here in San Diego with American Diabetes Association 77th Scientific Sessions. Today we have a unique person we will be interviewing. Instead of being an MD doctor, she is a PhD investigator that does research, so maybe we can get some inside scoops on the cure of diabetes.
Tell us a little about yourself and what you do?
Dr. Loeken: I am a basic researcher. I am very interested in understanding how normal cells function and what goes wrong when there are disease situations. Related to my research at the Joslin Diabetes Center, several years ago, when I was a new faculty member, I recognized that based on the work of Priscilla White who had been an early doctor at Joslin Diabetes Center soon after insulin was discovered, she took care of children with diabetes and also young women who now were surviving their diabetes with the availability of insulin. What she noticed when they were pregnant, it was particularly challenging to treat them and there was a large loss of life of the women with diabetes even during their pregnancies, so obviously even their fetuses would not survive either. By about the mid 1930’s, she had actually improved the care of women with diabetes enough that the survival of the women was very high. But, she noticed that there was high rate of birth defects in their offspring, so there was a high rate of perinatal and neonatal loss from the malformations. Now she made a lot of improvements in the care of women during pregnancy during her career, and she’s known among the specialists in diabetic pregnancy internationally for having really laid the groundwork for caring for women with diabetes during their pregnancies.
What I thought in the early ‘90s, a few years into setting up my own lab, was that the time was right for understanding on a molecular level what happens to the early developing embryo as a result of the mother’s diabetes leading to congenital malformations. Part of the reason why I thought this was a good time was because there had been a lot of genes that control formation of organ systems that had been identified from animals as far back as fruit flies but as recent as mice and there were methods to study how they might be regulated in a diabetic setting leading to congenital malformations. So, we set up a mouse model of diabetic pregnancy. And what we saw with very high frequency is one of the most common malformations in human diabetic pregnancy, which are neural tube defects. We also see some cardiac defects, which are also very common. We set out to study how it is that mother’s diabetes interferes with expression of the genes that would control the formation of the brain and the spinal cord and some structures in the heart. What is it about the increased glucose metabolism, as a result of the mother’s glucose going to the embryo that causes abnormal expression of genes. And really trying to identify are there particular pathways that we can say are crucial leading to malformations and might there eventually be strategies to prevent the malformations from occurring.
Steve: I know that you’re presenting here and I think the title is, XXX Our Way Into Understanding the Basic Science of Effects of Metformin and Glyburide on the Fetus and How That Affects Neural Issues. Your research focuses on birth defects caused by diabetic pregnancy. What kinds of diabetes can cause birth defects, and does gestational diabetes also cause birth defects?
Dr. Loeken: That is really a good question and it’s something that when I make presentations about I try to clarify at the beginning that we’re working with pre-gestational diabetes. Gestational diabetes, if it is truly gestational diabetes, has its onset about halfway through pregnancy. By that time the organ systems of the fetus have formed. While there still can be adverse effects on maturation of the organs, an increased risk for metabolic disease in the offspring later in the life, and also increased risk of fetal growth leading to birth difficulties with gestational diabetes, gestational diabetes does not cause birth defects. Either type 1 or type 2 diabetes can cause birth defects. What we think is that it’s the increased glucose circulating in the mother’s serum very early after she has gotten pregnant that then gets transported to the embryo and disturbs the molecular processes leading to organ formation.
Steve: I’m not sure that people have a real understanding. We know what pre-diabetes is. It’s an A1c of 5.7 – 6.4%. It’s kind of simple. When it comes to pregnancy, the limitations are much lower for obvious reasons. So, what is the definition of pre-gestational diabetes?
Dr. Loeken: It means she is not yet pregnant. When we say gestational diabetes, that is a form of diabetes that is characterized by more severe insulin resistance than is normal in pregnancy. It is only associated with the pregnancy and goes away after pregnancy. Pre-gestational diabetes means a woman is diabetic before she is pregnant, before gestation.
Steve: So, it is someone who has diabetes who is going to get pregnant?
Dr. Loeken: By the definition, if she becomes pregnant, then she is in gestation.
Steve: What kind of birth defects occur in the babies of mother with diabetes?
Dr. Loeken: Virtually any system that can develop a congenital malformation can occur with increased frequency in diabetic pregnancy. The most common are cardiac defects. There can be valvular defects. There can be outflow tract defect. The second most common defect are neural tube defects, primarily spina bifida. Although, anencephaly, which affects the head, can also occur. There can be renal malformations, some limb deformities, and other defects that are less common in pregnancy but are significantly increased in human diabetic pregnancy.
Steve: How important is it for someone before they get pregnant and during their pregnancy to maintain normal blood sugars. It could be very difficult, especially if they already have diabetes. What are the levels? What do you try to get them to do so that they have less chance for defects?
Dr. Loeken: Really the only strategies that we have right now are planning pregnancies so that a woman has taken measures. For example, taking folic acid, which is advised for any women who are planning pregnancy and also for diabetic women to try to get her glucose into good control as usually indicated by HbA1c levels. We do know that there are a lot of metabolic changes very soon after pregnancy occurs, even before a woman may even recognize she is pregnant due to the increased metabolism by the embryo and changes to mother’s own need for insulin. If she is in a good control of diabetes, if she knows how to control her diet, exercise, and insulin – if she’s taking insulin for type 2 diabetes, it may be insulin; it may be oral agents – but just to get her diabetes in as best control as possible to reduce the risks of malformations.
Steve: How has your research contributed to the understanding of how maternal diabetes causes birth defects?
Dr. Loeken: One of the things we showed with the mice is that high glucose levels seem to be teratogens and we did this by either only raising glucose levels in the mouse on a particular day of pregnancy, which actually corresponds in the women to about 4 weeks of pregnancy based on the last menstrual period or about the time she recognizes she is pregnant. Glucose above a certain level was sufficient to cause malformations. Also, in our diabetic model if we used a drug to cause excretion of glucose in the urine without changing any of the other metabolic disturbances of diabetes, that prevented the malformations. That shows that elevated glucose is essential and it is the cause of malformations. Another thing that we showed is it is known that there are families of transporters that are responsible for getting nutrients into the cells, including glucose transporters. Most of the glucose transporters throughout our body work very well when glucose levels are in pretty much normal range, 5.5 mmol, or 100 mg/dL. However, the embryo, in addition to expressing those glucose transporters, expresses the glucose transporter called Glut2, that is also expressed on pancreatic beta cells in the liver. Those tissues see higher glucose concentrations than our peripheral blood normally sees after a meal. Expressing Glut2 is very important for releasing insulin after a meal and storing glucose in the liver after a meal. We didn’t know at first why the embryo would express Glut2 because it would not function very well as the glucose transporter in non-diabetic pregnancies, but in the diabetic pregnancies it could make the embryo just like a glucose sponge, take up glucose really well when it is at high concentrations. We showed by using mice embryos that had mutations in the Glut2 gene – they either had one copy or zero copies of Glut2 – they were protected from the mother’s high glucose levels. That indicates that we know that Glut2 takes up glucose very well, when glucose levels are about 3-fold higher than normal, about 300 mg/dL or about 15 mmol. If we can keep glucose levels below those levels, you still may be getting more glucose in the embryo cells, but not be acting like the glucose sponge. So, I would say we want to strive not just to achieve good HbA1c levels, but monitoring glucose to keep those excursions away from the maximum uptake rate for Glut2.
Steve: What can diabetic women or their doctors do to reduce the risk that a birth defect will occur?
Dr. Loeken: So as I said, plan pregnancies, make sure that HbA1cs are in an advisable range, taking folic acid as would be advised for any women planning a pregnancy, and try to avoid unplanned pregnancies. But then just really be up on your cycles and if you might be pregnant to monitor your blood glucose frequently and try to keep the levels in a healthy range. You don’t want to be hypoglycemic either, and you certainly don’t want to avoid hyperglycemia.
Steve: Can you speak of the use of metformin and glyburide in pregnancy?
Dr. Loeken: Women may be taking either glyburide or metformin if they have type 2 diabetes, not type 1 diabetes. Many women, who are not necessarily diabetic but have polycystic ovarian syndrome, may be taking metformin with or without other drugs to try to improve their glucose tolerance, but also to normalize their menstrual cycles and their symptoms of polycystic ovarian syndrome. One of the reasons that we looked at metformin, and that’s the reason that I will be speaking about metformin here, is that one of the pathways that we’ve shown to be activated by high glucose in the embryo, leading to abnormal gene expression, is an enzyme that can be stimulated by metformin in the liver. So, we asked whether or not this enzyme might be stimulated in mouse embryos if you give their mothers metformin. And we gave them a dose that would be approximately the higher end of the dose that women might be taking. Although we did see that metformin may be stimulating this enzyme in the mouse mothers’ livers, we did not see any effect on the embryo. We didn’t see any stimulation of the enzyme and we didn’t see any malformations. What we think that’s due to is the fact that just as glucose has transporters that get it into the cells, there are transporters for metformin that get it into cells also. It looks like the early embryo, at least during the stage of development when malformations can occur, express very low levels of metformin transporters. So, they are not at risk, which is good. We know that having high glucose levels are increasing risks for malformations and we didn’t want to counteract the beneficial effects of increasing good glucose control by having a drug, metformin, that might counteract due to stimulation of the enzyme that high glucose regulates. So, it looks like metformin does not have that risk, at least in mice embryos. Obviously, you can’t do those same studies in human embryos. Looking at later pregnancy, there are still a lot of unanswered questions. Again. there are beneficial effects to the outcome of pregnancy from studies looking at metformin. Many women find is easier to take metformin, if they gave gestational diabetes or type 2 diabetes later in pregnancy, than insulin injections. But, those studies are still ongoing and I am not going to speak to recommendations there.
With regard to glyburide, so glyburide does not cross the placenta, not at meaningful levels. However, the embryo at the stage of development when malformations can occur doesn’t have a placenta, so glyburide could be exposed to the embryo. We know that the way that glyburide works is by binding to a protein that regulates the potassium channel, and that is expressed on pancreatic beta cells and not a lot of other tissues. The embryo at this stage of development doesn’t have pancreatic beta cells. So, if a mother is taking glyburide and becomes pregnant, I’m not saying that it is advisable, but we don’t have basic science evidence that there are cellular responses in the embryo that could be activated by glyburide.
Steve: We don’t do tests in pregnant women to see if all these other drugs are beneficial from what we know. Yet Sulfonylurea, glyburide, is approved for pregnancy and metformin. We don’t do a study with any drugs in a pregnant woman, yet these two are approved and we found them to be beneficial.
Dr. Loeken: There is a history of how one goes about testing drugs in pregnancy. In many cases there are preclinical studies that are performed with animal models and you try to do toxicity studies to see if there are any adverse effects. When I was setting out to do our experiments with metformin, I would say that the way that some of the other experiments were performed wouldn’t necessarily allow us to see endpoints for particular stimulation of the enzyme we were looking at and effects on the gene expression that we know can occur in diabetes. We felt that we needed to repeat those experiments, and at a dose that was close to a high end of a dose that would be given to women. Once there are safety concerns that have been overcome, the studies that have been performed have sometimes been done…well, I’ll give you an example. Some of the early studies that were done with glyburide in 1990’s or so were done with placentas after delivery where you can perfuse them. That is you can hook them up to a pump where there is glyburide going in and you look at what can come out through the fetal side. Where some of the older sulfonylureas could get through the placenta to fetal circulation, glyburide did not at meaningful levels. That then allowed studies where you would do in a random control insulin-treated women versus glyburide-treated women and look at the outcomes of pregnancy. Now I know of some studies, one of the other speakers at the same session that I’m at, Janet Rowan, has looked at comparisons of metformin and insulin in gestational diabetic pregnancies and she has been following the offspring of those mothers to see if there is long-term benefit. There have been beneficial effects of both insulin and metformin treatment, but there haven’t been enough of follow-up studies to determine the long-term effects on the offspring, which is one of the things we still think about.
Steve: Has your research led to any treatments or changes in diabetes care that could reduce the risk of birth defects in pregnancies of diabetic women?
Dr. Loeken: Our results support the recommendations of, for example, the American Diabetes Association and The International Association for the Diabetic Pregnancy Study Group that recommends planning of pregnancies, good glycemic control prior to pregnancy. What hasn’t necessarily been advanced yet is what can we do with technology to improve control of diabetes in pregnancy. Now, since the time I have been working in this field, I’s has been more common for women to use insulin pumps and to do continuous glucose monitoring. I think that our evidence that you need to have glucose in a relatively narrow range; you don’t want it to be getting close to the level where you’re going to be getting a lot of transport of glucose via Glut2, so if you can have a real-time sensing of glucose levels, I think that would be ideal. Even with our best technology, we don’t have the kinetics of insulin release, even from insulin pumps in the forms of insulin that adequately mimic what a normal functioning pancreas can do. So, I think that we need to get that technology, so that what the fetal circulation is seeing is more like what a normal non-diabetic pregnancy would be seeing.
Steve: Sometimes research doesn’t pan out for what you are looking for, but sometimes it opens the door for other types of research. In your research findings, from your lab, have any implications for other problems associated with diabetes been found?
Dr. Loeken: There are some of the same biochemical pathways that are activated in response to high glucose transport in embryo cells that also occur in other tissues that are affected by diabetic complications. We don’t know if the Glut2 transporter might be involved in any of those complications. If Glut2 is involved, if it is expressed in progenitor stem cells, or particular stages of regeneration, it may be involved in wound healing. So, that’s research that still needs to be done to see if the particular rates of glucose uptake via Glut2 could be involved in maybe some of the failure of other diabetic complications to improve. One of the things we have found, however, we know that a gene that we focused on that needs to be turned on for normal neural tube for brain and spinal cord development is inhibited by high glucose. We’ve shown that that’s due to a particular modification of the DNA that occurs in cells where the gene isn’t turned on and it fails to be removed when the gene needs to turn on. This is called DNA methylation, or it’s an epigenetic regulation. I think there may be DNA methylation effects involved in other diabetic complications that may be more involved in failure of the tissues once injured to repair themselves. But again further research needs to test that. One area though I think that could be useful in the future is most of the work we have done up to this point has involved our model of mice diabetic pregnancy and mice embryos, but for various reasons we have been using mice embryonic stem cells that we’ve derived under conditions that they can respond to high glucose levels like the mice embryo does, and we can form neuronal precursors from them, like the cells that would form the neural tube. We’ve shown that Glut2 seems to be very important for responsiveness of these cells to high glucose. But that the normal function of Glut2, seems to be, under normal glucose concentrations, to transport another sugar that then stimulates growth. If human progenitor cells, either induce pluripotent stem cells that you would make in the dish or are endogenous stem cells that are involved in, for example, wound repair in vivo, if they also express a transporter that could take up the sugar and lead to improved growth, this may give us more therapeutic strategies for other diabetic complications.
Steve: Technology has changed just about every aspect of what we do. When you leave here next year, an uber pulls up and there’s no driver and there’s a voice that says, “Step in. I’ll be your driver. You can’t see me, but I’m here.” I always ask people would you get in that car? [laughter] So things have changed dramatically, and what we have done over the last 25 years, we’ve actually duplicated and now we are doing it every 5 years. From your expertise, have defects from pregnancy caused by diabetes improved over the last years or are we still at the same place?
Dr. Loeken: I think we have a much better understanding, and we have data to support why women should be in good glycemic control. It used to be you’ll have a better outcome of pregnancy and now we know specific steps that occur in the embryo that will be normalized if you improve glycemic control. Technology, as I alluded to before, may allow us to use strategies to come more closely to giving glucose in the fetal circulation to what occurs in a non-diabetic pregnancy. That is not my work, but there might be artificial intelligence that is involved in continuous glucose monitoring and insulin pump that is a generation better than what we have now and is also user-friendly.
Steve: Would you say that CGM has made a major impact in preventing defects?
Dr. Loeken: I don’t think that there is good research in that. I do know of a few colleagues who have done some monitoring of that. For ethical reasons, you want to have women in good glycemic control. If you have women entering the study you want to get the birth defects down to as low as possible for ethical reasons. You need to have a large enough sample size so you can see a statistical difference when, for example, you have used a continuous glucose monitor over other non-continuous glucose monitoring approaches. I don’t think we have the hard data yet.
Steve: If you could have your druthers in your research, and be very successful, and win the Nobel Prize, what would you like to accomplish?
Dr. Loeken: I would like in this particular area to be able to say OK there is this one switch that we know causes a cascade of events in response to high glucose. If we can block that switch without having any other adverse effects, that would be an additional piece of security that we could give to women during their pregnancies. Experimentally, we do have some drugs that we can use that will block the effects of high glucose, but you wouldn’t want to treat a woman prophylactically with those drugs because they might have adverse effects of their own.
Steve: I see, if there is a pill that could prevent neural tube deficiencies, people would eat what they wanted and wouldn’t be so concerned with their blood sugars if they knew they didn’t have to keep them low.
Dr. Loeken: I am hoping the women will do the best they can, but as I say, our best technology does not get glucose levels to the embryo as close to normal as not having diabetes. So just that extra ability to provide the better environment to the embryo.
Steve: I want to thank you for your time, and enjoy the rest of your time in sunny San Diego and thanks again.
Dr. Loeken: Thank you.