#240 ‒ The confusion around HDL and its link to cardiovascular disease | Dan Rader, M.D.

Hey everyone. Welcome to the Drive Podcast. I'm your host, Peter Attia. This podcast, my website and my weekly newsletter all focus on the goal of translating the science of longevity into something accessible for everyone. Our goal is to provide the best content in health and wellness, full stop, and we've assembled a great team of analysts to make this happen. If you enjoy this podcast, we've created a membership program that brings you far more in depth content. If you want to take your knowledge of this space to the next level, at the end of this episode I'll explain what those benefits are. Or if you want to learn more now, head over to peterattiamd.com subscribe Now without further delay. Here's today's episode. My guest this week is Dan Rader. Dan is a Professor of Molecular Medicine at the Perelman School of Medicine at the University of Pennsylvania, where he conducts translational research on lipoprotein metabolism and atherosclerosis, with a particular focus on the metabolism and most importantly, function of high density lipoproteins, HDLs. Dan has received numerous awards and has been elected to the American Society of Clinical Investigation, the Association of American Physicians, and the National Academy of Medicine. He also currently serves on the Board of Directors of the International Society of Atherosclerosis, the Board of External Experts at the National Heart, Lung and Blood Institute, and and the Advisory Board for the Clinical Research of the nih. In this episode, we focus our entire conversation around high density lipoproteins, or HDLs. Now, as any listener of this podcast will know, we have no shortage of content around lipids, and we focus a lot of that energy on the apob side of the family, that is to some extent VLDLs and of course LP, which is a subset of LDL. However, this is the first time we're doing a dedicated podcast on hdls. Now, the reason for that, in other words, the reason for that disparity, is largely because HDL biology is so much more complex and we know so much less. I mean, at the highest level, I think people generally think of HDL as, quote, unquote, the good cholesterol. But you've no doubt heard me rail on the stupidity of such a designation. But there is clearly something good about HDLs, as in the lipoproteins, not the cholesterol. And in this discussion we we really talk about everything from the biology of the hdl, its genesis, its origin, its metabolism, its life cycle, and of course, its function. We also talk about why it has been so complicated to use pharmacologic interventions on the HDL side of the equation to impact atherosclerosis. Conversely, of course, it's been the easiest thing, I would say, in medicine, and perhaps the greatest success of modern medicine, especially as it comes to cardiovascular disease, has been our ability to manipulate the. The APOB side of the equation as opposed to the APO A side of the equation. That's one thing that's going to be important here. When we get into the terminology, when we talk about apo a in the context of HDLs, we're talking about apoa, as in a big A, which has no bearing whatsoever to APO little A, the thing that of course defines an lp. But I digress. That is simply one of the many details we get into. So, as I said, we're gonna talk here about the genesis of the hdl, the structure, its metabolism. We talk about the differences between HDLs, LDLs, the difference between the HDL measurements exactly as HDL cholesterol versus APOA concentration versus HDL particle number. Talk about the idea that having a high HDL means you don't need to worry about cardiovascular disease and how that's obviously going to be bunked. I'm just going to let the cat out of the bag on that one. We speak about CTEP inhibitors, which are a class of drug that have been repeatedly used to try to increase HDL cholesterol with the hopes that that would reduce cardiovascular disease. And finally, we end the conversation around some of the new thinking around HDL and neurodegenerative disease, something, again, I learned a lot about. So, without further delay, enjoy my conversation with Dan Rader. Well, Dan, thank you so much for making time to join us on the podcast today. Tom Dayspring really recommended you highly. And anytime Tom Dayspring says to me, you should have so and so on the podcast to talk about anything that has to do with lipids, I immediately pay attention.

Tom's an amazing guy.

Now, in particular, the subject matter I want to explore with you today is probably the area of lipidology that I personally am the least familiar with. And that has to do with kind of one half of the lipid family. You know, I explain this to patients as there are, broadly speaking, two families, the Apob family and the Apoa family. We spend, obviously so much more time talking about the APOB family based on, I think, two things. One is our clearer understanding of it, and two, the direction and causal relationship to pathology. But I often sort of waffle a bit when I'm talking about the apoa side of the house. That's really why I'm excited to be sitting down with you today. But perhaps for the person listening to this, who doesn't even understand what a lipoprotein is yet, never mind what the two families are that we're talking about, can you take it back to the beginning for where do these things fit into the broader architecture of our existence?

Lipoproteins are these big complexes that really are evolved to transport lipids within the blood. You know, lipids are like oil. They don't mix well with water. You know, oil droplets float to the top of a puddle. We wouldn't be able to transport lipids in the blood if we hadn't evolved a mechanism to do that. And lipoproteins are basically the mechanism to do that. As their name suggests, they're lipoproteins. They have lipid in the core, and then they have proteins that dot the surface that allows them to be transported in very complex and sophisticated ways within the bloodstream in terms of how they're metabolized, in terms of the receptors they bind to, and in terms of the specific proteins and lipids that they carry. You've talked a lot about apob lipoproteins. I've listened to several of those podcasts. They're really fabulous. And of course, the apob lipoproteins really primarily transport or evolve to transport triglycerides as a source of energy both from the gut to adipose and muscle and heart, as well as from the liver during times when we're fasting. You've talked a lot about that. And as you pointed out, they are very important in terms of causal relationship to atherosclerotic cardiovascular disease. Hdl, which we're going to be talking about today, is also a lipoprotein. It's a very complex lipoprotein. It doesn't have this key protein, apob. That's what differentiates them. That's why we often refer to the apob containing lipoproteins. And then HDL is the other half, as you said. And HDL really is characterized by a different protein called APOA1. HDL also transports lipids, especially cholesterol and some other complex lipids, and we'll be getting into that more. But essentially, lipoproteins are lipid transport vehicles within the blood and really evolved to do that function.

A couple things I'll just throw in for folks. Again, maybe new to some of this, which is all of this stuff we're talking about is so important in terms of these lipoproteins, because unlike glucose electrolytes, things that we kind of take for granted, that are water soluble, we use our circulatory system as the freeway to move these things around. And unfortunately, as you said, because of the insolubility of both cholesterol, triglyceride, lipids, et cetera, we have to come up with this more complicated system. I think the second thing I'll throw in just for folks, because it could get confusing with the nomenclature. When you talk about apoa, I want to make sure people understand we're not talking about lp, which has another apo, a little A. So maybe just clarify for folks the difference between those two because we'll do our best to not talk about apo little A today at all.

Yeah, apo little A is or LP is really important, not the topic of today's discussion. I think, as Peter already said, the main thing to remember is that that A is lowercase, whereas the A we're talking about today is uppercase. I know it sounds crazy, it's just the way things evolved historically. But we're talking about a apoa, uppercase a, and the most important one has a one after it. So apoa one. So you're going to hear us refer to apoa one a lot. Totally different than the lp, which is also an important topic, but not for today.

Yeah, it's a shame that the lipid community back in the 60s, 70s and 80s didn't hire kind of a PR firm to sort of help with the naming of this stuff, because it is, hands down, some of the most complicated nomenclature. And if they realized back then, I'm half joking, that it wouldn't be a bad idea for patients to actually understand this stuff. I think we could have done better. Okay, so you've already alluded to kind of different APOAs and APOA1. Again, that's APO, capital A, Roman numeral 1 is the most important. But let's go back even further than that. So again, not to draw the apobs into this again, but we kind of technically now have these two lineages of APOBs, right? We have the APOB 100. And virtually all of the time that we say apob, we really mean APOB 100. And that's to contrast it with APOB 48, the one that you kind of alluded to very briefly as transporting chylomicrons from the gut for the use of energy. Can you talk a little bit about the. Or not a little bit. Maybe A lot of bit about the genesis of the high density lipoprotein, what is its parent, where is it formed and how does it move through its evolution such that, you know, when we're measuring HDL cholesterol, something we'll talk about in a minute, we have a sense of where it came from in the same way that we understand VLDLs, IDLs and LDLs.

With all due respect to the APOB community, and I'm part of that community too, I would say the metabolism of HDL is perhaps an order of magnitude even more complex. And that's obviously what we're going to be working our way through today. So I'll start relatively simplistically and then Peter, I know you'll lead me to increasingly more detail about the process.

And by the way, I don't think anyone's going to disagree with that. Dan, meaning I don't think anybody who spent even a modicum of time looking at this literature, it is hands down the most confusing stuff in the lipid space.

Again, I'll start with Apoa1 as the key protein. With HDL, the analogy is to Apob, as Peter alluded to, although one big difference is Apob, as your listeners know, APOB stays with that particle throughout its lifetime. So it gets made by the intestine as B48 or the liver as B100. It stays with that particle. There's one molecule of this huge APOB protein and and that it basically then ultimately gets taken up mostly by the liver after its time in the blood. Apoa 1 is also made by the intestine and the liver. There's some parallelism there. It also is a core protein essentially of hdl. But unlike apob, there are several molecules of APOA one on any given HDL particle. We'll probably talk more about that, but anywhere from one to four, maybe in some cases a little bit more. And also APOA one doesn't stay with the particle. It can exchange onto other types of mostly HDL particles, but even sometimes onto APOB containing lipoproteins. What we've really learned in the last decade or so is that APOA1 is put into the blood by either the intestinal enterocyte or the hepatocyte in the liver as more or less a free protein. So it's secreted as a protein. One of the first steps that has to happen for HDL to be formed is that hdl, once it comes out of the cell, engages with a key transport protein called ABCA1. And Peter stop me if I'm already getting too detailed. ABCA1. We'll come back to that which its role. ABCA1's role is to take lipid, cholesterol and other types of lipids called phospholipids from the cell and export them to the newly secreted APOA1. So APOA1 has to acquire lipid, particularly phospholipid, and cholesterol soon after it's been secreted in order for the so called nascent or early HDL to start forming. We'll come back to this when we talk about genetics. But we know this because humans that lack ABCA1 have virtually undetectable HDL. And that is because they cannot, they make plenty of APOA one, but they cannot protect that APOA one with lipid once it's secreted and it goes out like a rocket from the blood, basically almost can't measure it in the blood. So that's the first step in terms of HDL biogenesis, if you will.

What other phenotype do those patients have? So in the absence of cholesterol accumulating in what would be an hdl, does it end up where more of it ends up in the LDL or back in the liver? Where does it go?

This is the value of studying these very rare human genetic disorders. And it just tells us a lot about what specific proteins or genes are doing this disorder, which we can talk about the history. It's a fascinating history. It's called Tangier Disease. It's named for a small, flat, bizarre island in the middle of the Chesapeake Bay where the first patient was discovered. This disorder is primarily associated with accumulation of cholesterol in macrophages throughout the body. So tonsils, spleen places where there are a lot of macrophages, the cholesterol really builds up and they have huge sort of orange colored tonsils. They have big spleens that sometimes rupture. They have big livers because there are a lot of macrophages and macrophage like cells in the liver. And they also have some neurologic type issues, especially neuropathy, which maybe we'll leave to later when we start talking about HDL and its relationship to the nervous system. They may or may not have some increased risk of atherosclerotic cardiovascular disease. But as we'll talk about that relationship between HDL and atherosclerosis is quite complex. This disorder has helped to inform that a little bit.

Another sort of structural question, Dan, if you were to take a quasi mature garden variety HDL particle. And again, it's more complicated there because of what we're talking about. And put it next to a comparable LDL particle. What's the difference in size? And can you explain a little bit why they come up with this different name of high versus low density? What specifically is that referring to?

That's a great question. Again, this is a historical artifact of how lipoproteins were discovered and named. First of all, HDLs are much smaller, I would say in the neighborhood of 1/5 to 1/10 the size of an LDL, and of course, much yet smaller than triglyceride rich lipoproteins. Lipoproteins have lipid, as I mentioned earlier, lipid floats. So lipids create buoyancy to particles. And the way lipoproteins were discovered is that when you subject plasma to spinning, the lipoproteins spin to the top. And they spin to the top at different rates under different forces. The low density lipoproteins have more lipid or bigger more lipid. They spin to the top more easily. The high density lipoproteins still have lipid. They do spin, but they don't spin quite as quickly or easily. So they're higher density than the low. And then in another artifact of sort of the history, Peter, after low density was discovered, something even lighter that spun up even more easily was discovered. And of course, if low was already taken, so that had to be called very low. And so there's the very low density lipoproteins that you've also talked about at length. And then finally the chylomicrons, the huge ones that come from the intestine. They're just so light and so buoyant that instead of calling them very, very, very low, they were just given the name chylomicrons.

How long does an HDL particle last? And does it mean the same thing, given that it's transferring its apoa in a way that makes it a little different from, as you said, the way the VLDL to IDL to LDL always stay with one lipoprotein apob that allows us to kind of track its life.

Before we get into kind of how HDL is removed from the blood. Could I take a little bit more time to explain what happens after that APOA 1 quote unquote nascent HDL particle gets formed? Because there's actually a bunch of stuff that has to happen first before we start talking about how the HDL then gets removed from the blood. So the APO one or what we call the nascent HDL has, you know, APO1, has phospholipids, and it has what I'm going to call free cholesterol. And this is a concept that you may or may not have addressed before, I can't remember which is cholesterol itself as a molecule can have a fatty acid attached to it. When you attach a fatty acid to a hydroxyl moiety on the cholesterol molecule, you create what we call a cholesterol ester. And the cholesterol ester is even more hydrophobic or oily than the free cholesterol because it has this additional fatty acid sticking off it, which is obviously oil. It's absolutely critical for HDL for the HDL maturation process to have this fatty acid attached to the cholesterol after the nascent HDL has been formed. And there's another key enzyme that is responsible for that, so called lecithin cholesterol acyltransferase. It's the last time I'm going to say that. I'm just going to call it lcat. That's its nickname, lcat. L C A T. So LCAT rides on the HDL particle, on that nascent particle, it takes a fatty acid from one of the phospholipids on the particle and it transfers that fatty acid to the cholesterol and creates the cholesterol ester. Remember, that cholesterol ester is more oily. It actually then moves to sort of the center of the particle because it wants to be by itself and not interacting with the water, and it starts to form the core of the HDL particle. So the core of the mature, what I'll call the mature HDL particle is basically cholesterol esters that are entirely dependent on LCAP for their formation. Another rare condition are people who lack LCAT genetically. And as you might maybe expect from what I just said, people who lack LCAT who can't do that process of esterifying the cholesterol have extremely low levels of hdl, not quite as low as Tangier disease, but very low, and simply can't form the mature HDL particles that we see in most people. So again, we learned very important things about the key role of that enzyme that ultimately leads to the mature particle. That then is really what we're measuring as HDL cholesterol in people is this mature HDL particle where most of the cholesterol we're measuring is the cholesterol ester in the core.

Just to make sure we're clear on semantics. When you say that those folks have very low hdl, you mean low HDL cholesterol or a low concentration of APOA1 or any of the APOAs.

They have low HDL cholesterol. So again, Tangier disease, HDL cholesterol is one virtually undetectable LCAT deficiency. HDL cholesterol is 10 also, as we'll talk about, quite low compared to normal. They also have low APOA1 levels. And part of that is because part of the metabolism of Apoa1, which is where you were going, is that it's affected by the mature HDL and the cholesterol in the core. So when you don't make that cholesterol ester, that APOA one is much more rapidly removed from the blood. It's not the normal metabolism. And basically the kidney sees that APOA one, which doesn't have much cholesterol on it and actually filters it and degrades it. Not surprisingly, the biggest issue with LCAT deficient patients is they get renal disease, serious progressive chronic kidney disease. In these patients. That leads usually to kidney transplant related to something around the lipid metabolism in the kidney as a result of the lack of LCAT activity and esterification of cholesterol.

Before we go deeper on this, I just want to ask if there are any insights we have or how parallel is this system in other mammals, whether it be primates, you know, dogs, mice, et cetera, because obviously a lot of these animals have a very different APOB side of the equation. I'm curious as to how similar they are on this front.

No, it's a great question. Apoa1 is pretty highly conserved among certainly among mammals. In many lower level mammals, the primary lipoprotein is HDL. That clearly includes APOA1. I'll just take mice, obviously a major model system for human, for biomedical research, mice. I'd say 90% of cholesterol in the blood of mice is in HDL, even more than 90% in a normal mouse. That is the primary lipoprotein in mice. It's true for dogs, it's true for really all mammalian species. I mean, once you start getting below mammals, things change a little bit in terms of the lipoprotein metabolism. Probably we won't get into that today, but there's even HDL type particles in much lower species leading to one of the kind of thoughts that HDL is kind of like the primordial lipoprotein and that the APOB system kind of evolved subsequently, but that HDL probably Very early evolved as a transport mechanism for lipids within circulatory systems in lower level species.

You know, this to me is one of the most interesting kind of teleologic evolutionary questions, which is, was God just pissed off at us? And is that why we as humans have apob? Because I can't. I've never really heard a convincing explanation for why humans have APOB when it seems that every other species gets away without it. Right, as you said, I mean, if a mouse or a primate or a dog, who clearly have similar energetic requirements, can use their APOAs, I.e. hDLs, for lipid transport and energy mobilization, why did we need to evolve with this particle that in the short term really doesn't cause us any trouble, of course, but long enough it doesn't. And so one answer might simply be that evolution had no concern for the longevity of our species.

That's for sure.

But there is some other short term benefit from APOB that maybe outweighs the long term death that it brings our species via ascvd.

I do want to be clear. Mice, dogs, pigs, rabbits, they all make APOB in both the gut and the liver. So it's not that these species don't have apob. The difference is, in terms of looking at the steady state plasma levels, is that their APOB system is just really markedly revved up. They take care of their business, they.

Just must clear it out of a certain place and they make it so much more.

And it serves a similar role, you know, in terms of transporting triglycerides as a source of energy to tissues in the body that need energy and to adipose tissue for storing it. It's just that the rate at which they do that is much more efficient. Whereas in humans, for reasons we could talk about, but aren't totally clear, but certainly have to do partly with our lifestyle, humans are not nearly as efficient at clearing the APOB containing lipoproteins, and they hang around, especially as ldl as a byproduct of this metabolism in a way that then ultimately leads to, of course, atherosclerotic vascular disease. We do need apob. Honestly, mammals evolved APOB because one, we have to be able to efficiently capture fats in the diet for energy purposes very efficiently. That's what the chylomicrons allow us to do. And during fasting, which of course in evolution sometimes lasted a long time, we have to be very parsimonious about parceling out that available fat we have in our adipose stores back out to heart and muscle to be able to have as a source of energy. That's what APOB 100 in the liver does. We need these, especially in evolution, the latter, we need it very substantially. But now, unfortunately, in the modern times, the apob system is mostly a problem, not something we need.

And I think the way you stated it earlier is obviously more accurate and perhaps I want to just make sure I'm restating it without being quite as tongue in cheek. It's really not that we don't need apob, it's that we could be clearing it at a much greater and more efficient rate than we are. And we could more mimic primates and other mammals and walk around with an apob concentration of, call it 20 milligrams per deciliter instead of 100 milligrams per deciliter. A five fold difference, which is about. That's the difference between getting atherosclerosis in our lifetime and never getting atherosclerosis in our lifetime.

I agree with that.

And that's the interesting thing because even absent kind of the quote unquote lifestyle, things that will raise apob, it just occurs with aging. You know, Peter Libby has written about this very eloquently and children, neonates have apob concentrations that are just like those of these other animals. And all things equal, as we age, apob just goes up, that concentration goes up. As you say, we're less efficient in the way we clear apob from circulation. Anyway, it's a fascinating topic and it's. I know not what we're going to talk about today because it's one of speculation and we could speculate, but there are actually lots of things today that we don't have to speculate about. As it comes to apoa and hdl. I want to go back to kind of the nomenclature a little bit because I know I'm still kind of confused on some of this and I think it's going to be a little challenging because we're sometimes talking about Roman numerals and we're sometimes talking about numbers. But is there just an APOA 1 and 2 in the Roman numeral APO lipoprotein cover?

No.

So it goes to 2, 3.

The two other APOAs that at least we know about or we talk about are APOA 4 and APOA 5. Don't ask me what happened to APOA 3. That's a mystery I've never been able to solve. But anyway, it's another one of those APO4, we don't really know what it does APOA5, I can't remember if you've talked about, but APOA5 has a very important role in stimulating lipoprotein lipase and in the metabolism of triglyceride rich lipoproteins. It rides on hdl, but it also rides on triglyceride rich lipoproteins. And people who lack APOA 5 have really high triglycerides and are also at major risk for atherosclerotic vascular disease. So, anyway, again, maybe not the topic for today, but it does illustrate one point, Peter, maybe that I'll make, which is that HDL as a molecule, as a lipoprotein, I think of as a platform. It's a platform for all sorts of proteins and lipids that get transported by HDL in the blood and then transfer off HDL to other things to APOB containing lipoproteins. So my example of Apoa 5, it's named Apoa because it was discovered on HDL, but its primary role in metabolism is triglyceride rich lipoprotein metabolism. APOA5 is basically kept within the blood and stored, if you will, on hdl. But then when we eat a fatty meal, it transfers off to the triglyceride rich lipoproteins and it serves a role of promoting the hydrolysis of the triglycerides. So I think that's a general concept for HDL that maybe we'll keep coming back to is this platform for all sorts of things that are doing different things that involve not only lipid metabolism, but host defense and other things that it evolved to do as a platform for transporting things within the blood, which.

I think explains, at least in part, the complexity of this system is it's much more dynamic than what we see on the APOB side. The APOB side, as you said, it's a very monogamous relationship, right? I mean, you sort of get your APOB in the liver and it's with you for life and you're not swapping, you're not trading, you're not increasing or decreasing, you're just marching through life. And the HDL is maybe the monogamy wasn't the best example because now I don't know what to come up with here. Because it's not just polygamy, it's even more complicated than that. As you said, it's swapping, it's moving things around, it's carrying things that it doesn't really use, but loaning them out. It is indeed a complicated system. The next thing that seems to add a lot of confusion, at least to me, is some of the nomenclature around the numbering of the HDL particles. And I bring this up in part because various commercial labs, someone listening to this goes out and gets a really fancy, fancy blood test. We use Boston Heart labs with our patients. I know that this is something you see with a number of other labs. It might say things like HDL1, HDL2, HDL3. What's the relationship between that nomenclature and the APOA1, APOA2, APOA4, et cetera.

This is ridiculously complex. The short answer to your question is actually, believe it or not, there's no relationship. So the numbering of the apolipoproteins or the Apoa lipoproteins 1, 2, 4, 5 is totally independent of the numbering of the HDL particles, like especially HDL 2 and HDL 3, which are the classic two main subclasses of HDL. These are different sizes and different densities of HDL that again were isolated by centrifugation and by their flotation properties. HDL2 is bigger, HDL3 is a little smaller. HDL2 has mostly 4 molecules of Apoa1, HDL3 has mostly 3 molecules of Apo1. But those threes don't have anything to do with each other. And then to make things more complicated, there are other ways of sub fractionating lipoproteins that I know you've discussed, like the NMR type methodology that don't use HDL2 and HDL3, which frankly is a little outmoded now, but use things like very large hdl, large hdl, medium hdl, small hdl, very small hdl. HDL certainly comes in a whole series of sizes and densities that can be isolated by these different methodologies. There's been a cottage industry, as you know, Peter, of trying to relate these different fractions of HDL to cardiovascular risk in a way that might give us some advantages in terms of trying to predict risk. We can come back to that. But I would say in general I'm not very compelled that fractionating HDLs gives much clinically valuable information that allows us to predict risk. In contrast, I just want to say in contrast to the APOB containing lipoproteins, where, whether it's APOB or whether. But I think the smaller, denser LDL particles do have a relationship to increase risk, as you've discussed in the past. But I think the HDL fractionation is fascinating for understanding HCL biology and metabolism, but is relatively unimportant from A clinical relevance standpoint.

I'm actually glad to hear you say that because I was wondering if I need to be changing our clinical practice. In about 2016, we basically stopped paying attention to any of the HDL fractionation metrics and really had to go out of our way to make sure that the lab was not running those because in some areas we're running complicated labs and we were sort of saying, look, we don't want any of this stuff A we don't want the additional cost of it. But more importantly, we don't think it's clinically actionable. We still get labs from other doctors who might have seen our patients in the past and they're chock full of these things to which I don't know what to do with them.

But while we're on this topic of HDL fractionation, unless you want to ask me another question.

No, no, go ahead.

I wonder if I could use this as a springboard to continuing this complex saga of the metabolism of HDLs once they're formed and once they have cholesterol ester. And the reason we have this whole sort of set of different size and density HDLs is because of the complex metabolism that occurs on the HDLs once they're in the blood and once they've kind of been made mature HDLs. I want to give you maybe a couple examples of that. One is that HDLs are acted upon by so called lipases and you've talked a lot about lipoprotein lipase. It's critically important for triglyceride metabolism and energy metabolism. Lipoprotein lipase has two other very close cousins that I don't think you've talked nearly as much about, appropriately because they're more HDL related. One is called hepatic lipase and what is called, quote, endothelial lipase. We actually first reported a long time ago endothelial lipase as a member of this family. So both of these lipases, they're made in different places as their names suggest. But both of them fundamentally chew on hdl, specifically the phospholipids on HDL and result in modification of the phospholipid composition of the HDL particle in a way that does different things. It certainly changes the sizes of the HDLs and they contribute to this distribution of different sizes. It changes the protein composition of the HDLs in ways that we don't fully understand and almost certainly has other important biological effects that we don't fully understand. But of course, these evolve for some reason, one of which I'm going to get back to later when we talk about the central nervous system. So that's one lipases are really critical for HDL metabolism. There's also this protein that I know you've had some discussion about, cholesterol ester transfer protein. Happy to go into more detail, and I'm sure we will because it's very relevant to hdl, but just for the metabolism part of it, this so called CETP cholesterol estro transfer protein essentially transfers cholesterol esters between APOB containing lipoproteins and hdl. And it's a major modifier of the HDL particle in terms of its size and composition. We know this because people who lack CETP have hugely elevated HDL cholesterol levels big time, like way over 100. That really told us that cholesterol ester transfer protein siphons cholesterol out of hdl and when you don't have it, you have a lot more cholesterol in hdl. And just point back to our previous discussion of mice. Mice don't have ctp. So among the differences, one of the things is that mice have a lot more HDL cholesterol relative to apobio because they lack this CTP protein.

I mean, I know we're going to come back to this, but I just, I worry that for the listener, this would be an opportunity missed if we don't dive into CTEP a little bit because of the following. If we take a step back, a listener is going to be saying, peter, Dan, what the hell are you guys talking about? Like, I am so lost. The only thing I know is when I go to the doctor, the doctor says, my good cholesterol is high, I'm in good shape. So let's do this. Let's hit pause for one sec and acknowledge that a standard lipid panel spits out a bunch of numbers. Total cholesterol, LDL cholesterol, HDL cholesterol, and if you're lucky, VLDL cholesterol, non HDL cholesterol, and triglycerides. That's basically the standard metric. If the lab is competent and they're using direct measurements, your HDL cholesterol, LDL cholesterol and VLDL cholesterol should sum to your total cholesterol. And of course, your non HDL cholesterol should be the same number as your total cholesterol, less your HDL cholesterol. Want to pause and also insert that with nomenclature? HDL is not a laboratory metric. It is a lipoprotein. The laboratory metric is HDL cholesterol, HDL CD, or if using NMR, HDLP, HDL.

Particle number, or APOA1. An analogy to measuring APOB.

APOB, exactly. The units that you've been throwing around, of course, are the HDL cholesterol. So a moment ago you said, hey, people who are deficient in ctep could have HDLs over 100. Of course, that means HDL cholesterol over 100 milligrams per deciliter. Now, there is an observation that goes back probably to the late 70s, right? I mean, it probably goes back to late 70s, early 80s, which is in some of the earliest Framingham cohorts, which observed the risk of ASCVD in five cities. We don't need to go into what Framingham did in the first cohort, but what came out of that was higher HDL cholesterol was better than lower HDL cholesterol. In fact, that was four times greater, if my memory serves correctly, as a predictor of ascvd than high LDL cholesterol was a negative predictor. Am I remembering that correctly?

Yeah, that's about right. Yep.

So it's by now we're talking about 82, 83, 84. This has been a long time since I've looked at this stuff, but it emerges that, hey, high HDL cholesterol seems to be positively associated with outcomes. And obviously that's a big part of why HDL cholesterol became known as good cholesterol and LDL cholesterol became known as bad cholesterol. We'll obviously talk about why those terms are inaccurate. But it wasn't long until companies, drug companies were saying, hey, wait a minute, we know that if CTEP is inhibited, this enzyme is inhibited. HDL cholesterol goes up. That must be a good thing, right?

That led directly from this observation I mentioned that people who lack CTP genetically have these hugely elevated levels of hdl, one directly from the other. Well, gosh, that must mean that if we could pharmacologically inhibit ctp, it would be a way to raise htl. And of course, as you know, that absolutely turned out to be the case. But the story goes beyond that.

So the first company to do this was Pfizer. Right. Wasn't the first CTEP inhibitor.

The first CETP inhibitor, torcetrapib, was from Pfizer. Correct.

And that was, you could take a skeptical view of the trial, which was Lipitor was about to go off patent. And so they came up with a trial that was Lipitor by itself versus Lipitor combined with the CTEP inhibitor. And I remember actually this was probably early 2000s, right? Thinking, oh, this is super. I mean, this is, you know, back when I was in my surgical residency. So I was only tangentially interested in this. Right. It wasn't my field, but interesting in that I knew my family history for ASCVD was high, so I was paying attention. And I remember thinking, well, God, this has gotta be great, right? You got one drug that's gonna lower LDL C, one that's gonna raise HDL C. This is a can't miss drug. And then In September of 2006, lo and behold, not only did it not get better, it was slightly worse. Right?

Yeah, bombshell. So first I wanna emphasize the genetics did predict what happened. That is pharmacologic inhibition of CTP is extraordinarily effective at raising HDL a lot. In fact, putting people kind of over 100 HDL cholesterol like the folks who lack CTP. But you're absolutely right. A lot of excitement about that, A lot of excitement about HDL is the good cholesterol. And that trial, the first of several with different CTP inhibitors, not only didn't show benefit, but showed an actual adverse effect. Now, it has to be said that in retrospect, we're pretty sure that adverse effect, more people, literally more people died. That adverse effect was due to off target effects of the drug, not due to CTP inhibition itself. So that drug of course was discarded, but it didn't kill the field because the idea was, well, that was just a bad drug. Let's get a clean CTP inhibitor and see what that really does. I don't know if you want to ask me questions or I can continue the story.

Yeah, let's go down that path because it's still a little. It got progressively less murky as time went on. But let's go down the path of the evolution of CTEP inhibitors, which, let's be honest, it's for what we're almost actually 16 years post, the halting of that trial and the discarding of that drug. And we still don't have a CTEP inhibitor on the market, Right?

We don't. There is still a CTP inhibitor that's in development, which I'll get to, but there is certainly not one on the market, as you know, Peter, and certainly probably many of our listeners do. Three additional CTP inhibitors were then taken into late stage clinical development, including large cardiovascular outcome trials, to summarize the results of that. One of them was just flat didn't do anything, didn't hurt people, but didn't help. A second one was stopped early because it really didn't look like it was doing anything helping. And the third one was followed through and did show about a disappointing 9% reduction in cardiovascular events. But I also want to point out that it lowered LDL and APOB pretty well also 9%. Given that you also lowered LDL and APOB was not exactly exciting and that drug was not taken further in terms of approval. I have to say one other thing that's just fascinating for sort of our field and maybe in general, one of these drugs, Dalcetropib, that failed in its clinical trial, a detailed genetic study was done post hoc and there were individuals who were found to have a particular genetic variant that looked like on post hoc analysis, that group actually benefited from the drug. And that led to a subsequent attempt to do another trial focused specifically on people of that genotype with this CETP inhibitor, which was just reported out really within the last year as a negative trial. That CTP inhibitor actually had two different trials, one in kind of a very focused precision medicine way, but it still didn't produce any benefit. This has been a long saga of using CTP inhibition to raise HDL as a way to reduce risk. And as we're going to be talking about more, it really is one of the key planks that has led to, I think, what is now rock solid, which is HDL cholesterol itself. The HDL cholesterol itself is not directly and causally protective against atherosclerotic cardiovascular disease. There's a lot of nuances there which we'll come back to, but I feel pretty confident making that statement.

And I just think this is a great opportunity to also talk about why it's so important to have hard outcome trials in the field of cardiovascular medicine. I think it's true in all medicine. But let's go back and think about the first lipid lowering drug introduced in the United States In, God, about 1950, right? Maybe 1959, something like that. Right. So try Panerol, which I can never remember the name of the enzyme, but it's the enzyme that converts desmosterol to cholesterol in the cholesterol synthetic pathway. Believe it or not, this is a sign of my aging, Dan. I used to know the names of these enzymes. It was like, and it's not Delta 24 desaturates, but it's a cousin or derivative or Something. Right. But anyway, so you had this drug tripyranol that inhibited that enzyme and lo and behold, it lowered cholesterol. Now this was for the listener. This is back in the day before we knew anything about the sub fractions of cholesterol. So we just knew from some of the early work of Ancel Keys that if you looked at very, very high levels of total cholesterol and compared that to people who had very, very low levels of total cholesterol, there was a difference in outcome, cardiovascular outcomes. This was an observed finding. There was no intervention to test that. So this drug came along and it really lowered total cholesterol and it likely would have been lowering LDL cholesterol and apob. It basically got approved on the basis of that, without of course, the heart outcome. So it gets approved, it goes into circulation and it's really only after it was approved, gosh, probably what, eight to 10 years later, that they had enough post surveillance data to say, you know what, this drug is lowering cholesterol, all right, but it's actually increasing mortality, cardiovascular mortality. The drug was pulled off the market. I don't know what is known about the why, but I know Tom Dayspring and I have speculated that it was probably the Desmosterol spike that was causing the problem. So you might have been trading one problem for a worse problem. Are you familiar with that drug or that story?

Only peripherally, but of course there's been a ton of research in Desmosterol really over the last several years. We now based on that, the plausibility of the fact that the adverse effects were related to increasing Desmosterol I think is quite real. That very well may be the explanation.

By the way, this is a totally tangent, off topic point, but it's become quite in vogue to use Clomiphene or Clomid for testosterone replacement in men. And the reason for it is a, it's quite effective. So if you give Clomiphene, you are telling the pituitary to make a lot of LH and fsh, which of course is telling the testes to make a lot of testosterone. This would certainly in the short term have the advantage of, of preserving testicular function, unlike giving exogenous testosterone which suppresses testicular function. And I think for short term use it probably isn't a bad thing. It's an off label use, of course, for clomiphene and we used to do it for this purpose. Right. So if a guy was still considering reproduction or we were considering this, a bridge treatment, between testosterone we would use it. But because we always measure Desmosterol, composterol, Sitosterol, these sterols, when we measure our patients cholesterol levels, we noticed how high the Desmosterol levels were getting in those patients and we figured out it was pretty quickly it was the Clomid that was doing this and it would reverse. But it could take a year to return desmosterol levels to normal after you stopped the Clomid. So about four years ago, once we put two and two together, we stopped using Clomid. But it's interesting to me that the use of that hormone has gone through the roof. There are now like Clomid clinics opening up and I don't understand why someone. And we've done a lit search. I don't understand. We just need to write this up. I think at some point. Yeah, it's fascinating because I don't understand why someone hasn't put the two and two together, especially long term use. Again, I don't think using Clomid for a couple of years is going to be problematic and I certainly don't think it's problematic for women using it for ivf. When I think about a guy being on this for 10 years and I'm talking desmosterol levels going up by 20 fold, that would be concerning. And we went back and tried to see if we could find out how high the desmosterol levels were in the tripyranol trial, not trials, but in the levels that if anybody had pulled serum or saved serum from those subjects, we couldn't find it. So we don't really know how high it was. But the assumption has to be that that drug and Clomiphene were both interfere with the same enzyme.

Really fascinating. I would encourage you to write that up.

So what's the point of that whole long winded story? The point of that long winded story is things can make a lot of sense until they don't. Right?

That's for sure. Before we leave CTP inhibition though, I do want to just remind your listeners that there is still one CETP inhibitor, obacetrapib, that is still in clinical development. What differentiates it? It's much more effective at lowering LDL and apob. We now no longer think that raising HDL cholesterol with CETP inhibition is gonna help you. We don't think it hurts you, but we don't think it's gonna help you. But we do think that there's still merit. Of course we know there's merit to raising LDL cholesterol in apob.

You mean lowering.

I'm sorry, rate lowering. Perhaps the CTP inhibitor could be part of the armamentarium to do that. So we'll see. Stay tuned.

Now, Dan, maybe this was naive of me, but after the third failure of a CTEP inhibitor, my very crude interpretation is not unique. I'm sure a lot of people have speculated this is that if at least part of the benefit of HDL involves stuff we're gonna talk about, right? Delipidation, reverse cholesterol transport, all of those things, and you slap a CTEP inhibitor on one of those particles, thereby making it harder for the particle to efflux its cholesterol to get rid of its cholesterol, which is why you now measure much higher HDL cholesterol, which on the surface looks good. That could actually be problematic. In other words, if you see a lot of people in a room, it might be tempting to conclude that there's something awesome going on in that room. But what you don't know if you can't measure all the ins and outs, is it might be that those people are all stuck in the room because the door is locked. I know that's a bit of a crude analogy, but is there any merit to that sort of thinking around the different ways in which one might boost HDL cholesterol and how some of those could actually be deleterious if they prevent function?

There absolutely is, but I think it's probably not relevant to CTP for reasons we can return to. But it is relevant to another protein that I think now's a good time to introduce, which is another key protein, a receptor that is basically the major HDL receptor, essentially the equivalent of the LDL receptor, but for HDL, and it's known as SRB1. Another unfortunate nomenclature, but SRB1 is a receptor that basically is on a lot of cells, but the liver is the most important with regard to hdl, and it essentially binds the HDL particle, basically sucks the cholesterol ester out of the HDL particle and then releases the cholesterol depleted HDL back into the circulation to go back around and do whatever it's doing. So the analogy I like to use, Peter, is that of garbage trucks. So a very simplistic view of hdl, which we're going to return to because it's much more nuanced, is that HDL to a certain extent functions like garbage trucks that are picking up things, trash, in places where you don't want it, and returning it to the liver, dumping it off via S or B1, and then going back now empty, to do more of its role again. It's more complicated than that. But that's an analogy. So this brings me back to SRB1. So there are humans who lack SRB1. What do you think the problem is?

Very high HDLs.

They have very high HDLs. They have very High HDLs because they can't unload the dump trucks, but they actually have increased risk of heart disease because they are not efficiently unloading that hdl. So very similar to your analogy of the locked room. You're not clearing the HDL and doing that normal process of recycling the trucks back to the periphery to do what they need to do. And so that's, I think, our best example of this concept of constipation of the system leading to high hdl, but paradoxically, to increase risk. And, of course, the analogy would be that. Not the analogy, but the interpretation. That would be you would never want to develop an SRB1 inhibitor. Yes, it would raise HDL quite a lot, but it wouldn't protect against heart disease and probably would hurt people.

Yeah. I remember the first time Tom shared a case study with me of a patient with presumably defective, not necessarily completely absent, but SRB1. And this was one of those things where you could tell just reading the journal, it must have been 40 years old. But her LDL cholesterol was, whatever it was, 100 milligrams per deciliter. Her HDL cholesterol was 150 milligrams per deciliter. So she had, quote, unquote, high cholesterol. But initially, people assumed it was not of concern because the fraction that was HDL cholesterol was what was contributing to it. And of course, on further exam, I mean, she had very, very advanced atherosclerosis for a woman her age.

Yep, exactly.

We don't see this often. I can tell you that. I have not yet seen a case of that. Now, admittedly, my practice is not large, but we're certainly looking for it when we see people with high HDL cholesterol, typically, you know, north of 90. I guess we should talk about this as well. But we can do that in a moment after this question, which is, why is there a sex difference between these two? Because there is quite a significant sex difference between men and women in terms of HDL cholesterol. But at what point, Dan, as a clinician, do you start to worry about that? And if there are doctors or patients listening to this, when Would you recommend that somebody go and get checked out for a genetic deficiency there?

SRB1 deficiency is not common. I completely agree with you. A huge project we've had for a long time now is essentially collecting people or consenting people with extreme high HDL with the goal of trying to find what the underlying genetic cause is. And I will tell you that that's how we found a few of these SRB1 folks. But most of them don't have any identifiable gene that we can point to and say here's the smoking gun, here's why your HDL is high. HDL is very heritable, meaning there's a lot of genetic determinants of hdl, but it's more about so called polygenic inheritance where multiple different genes, including SRB1, but not major genetic defects, just sort of more common variants. But many others, like some of the things we've been talking about, including SCTP and ABCA1, all contribute to the heritability of the HDL cholesterol. But to get back to the clinical implications, you know my view at this point we can talk more about the data. It's pretty clear that high HDL is not uniformly associated with protection and there are certain circumstances where that's true, especially if it's extremely high. Perhaps we'll get back to this, but there's a fair amount of evidence now, including a recent paper, that individuals of African ancestry who tend to have higher HDLs anyway, that the high HDLS are not as protective or maybe not even HDL is that well associated with risk. So my clinical advice to anyone with high HDL is not go get yourself sequenced to find out if you're deficient in SRB1. The odds of that are extremely low. But never use a high HDL as a reason for not using a statin or some other LDL lowering or preventive therapy. That is you should never be dissuaded from doing what you would have otherwise done in that patient just because their HDL is high. Does that make sense?

It absolutely makes sense. And I have a very short paragraph in my book that'll be coming out in a while where I cite two Mendelian randomizations that really look at this in some detail. I think I've explained Mendelian randomization before on the podcast, but it's always worth, I think, a short explanation. Again, in case you haven't heard it, this is basically a technique where you look for Genesis that impact traits that you are interested in. So in this case, because as you pointed out HDL is very genetic. That means that there are sets of genes that we would identify that predispose people to having very high versus very low HDL cholesterol. And because those genes are randomly occurring, you can look at that as though it's a natural experiment. And you can look at, based on the natural occurring, spreading or scattering of those genes, how are outcomes affected? And it turns out that low HDL cholesterol is not causally linked to atherosclerosis, and high HDL cholesterol, genetically high, is not causally linked to protection from ascvd. So I think those are very important findings, and I think it speaks to why, as you say, you can't use high HDL cholesterol as a reason to not treat in the presence of other risk factors.

I completely agree.

It's interesting, though, that that persists, isn't it? I do find this to be probably top three most vexing discussions I have with other physicians, which is, I know his LDL cholesterol is 140 milligrams per deciliter, but God, I mean, his HDL is 80. I mean, you know his ratio. I mean, that's when they say the ratio of total cholesterol to HDL or LDL to HDL is such and such, and therefore I don't need to treat. I mean, it makes me wish I had hair to pull out.

You're absolutely right. And I'll just reiterate, high HDL is never a reason not to treat someone who would have otherwise merited treatment. I do want to make clear, though, there are lots of people who are on the fence about whether to start a statin who have LDLs that are kind of borderline or even not that terribly high. And of course, we look at the whole patient, we look at the risk, we look at their calculated risk, other risk factors. But I'll just say that a low HDL in the setting of someone who you're generally on the fence about treating perhaps could contribute to tilting toward, yes, I'm going to treat this person. In other words, a low hdl, at least in most populations. Again, I think individuals of African ancestry, perhaps we have to be a little bit more careful about using HDL as a predictor. Maybe we'll come back to that. Otherwise, I think a low HDL can be used not absolutely, but relatively to tilt toward being more aggressive, but only in the context of the overall risk profile.

And obviously part of that, Dan, has to do with the relationship between low HDL cholesterol. If my Memory serves me correctly, it's the HDL 2 fraction and the association with that and insulin resistance. So there's no question that a phenotype. And as you point out, and we should come back to this, we don't even do this calculation for African American patients because we've long observed it's not helpful. But in non African American patients, the ratio of triglyceride to HDL cholesterol when both are in milligrams per deciliter is reasonably associated with insulin resistance. And the higher that ratio, the more insulin resistant they are. And obviously that ratio is driven up by an increase in triglycerides and a reduction in HDL cholesterol. Why is it that. I mean, we spent hours on this podcast talking about why insulin resistance would lead to or be associated with high triglycerides. We haven't done the opposite or the reverse of that. What is it about IR that drives down HDL2?

I'll just say first though that one of the other analogies I like to make is that HDL cholesterol, while not causally related to disease, is sort of like an HbA1C for cardiovascular risk factors. It's an integrator of information related to insulin resistance, related to triglycerides, related to inflammation that in one number, in most people, when it's low, it's telling you something about cardiovascular risk, even though itself it isn't directly impacting on risk. So in answer to your question, I think one of the big issues with HDL cholesterol is that it's in inverse barometer of triglyceride, efficiency of triglyceride metabolism. And again, we talked about efficiency earlier. You can only learn so much from measuring a fasting triglyceride after a 12 hour overnight fast. It's useful, it's the way we do it in terms of lipid panels. But a lot happens after a fatty meal and a lot of that action is basically over in most people by 12 hours. Some people are extraordinarily effective at clearing their dietary fat and even their liver derived fat, like mice. Others are not so effective, but their fasting triglycerides may not necessarily even reflect that. The HDL cholesterol does reflect that. There's a complex, as we discussed earlier, complex frequent interaction and exchange between triglyceride rich lipoproteins and hdl. With the net effect being the higher the triglycerides at any given time, the lower the HDL is as a result of the complex interactions. So if you can picture we do a lot of these experiments where we bring in people and we give them a high fat milkshake, we draw blood multiple time points after that milkshake, and we measure triglycerides and all sorts of other things. People differ a lot in their response to that high fat milkshake challenge. The higher that triglyceride goes, the area under the curve, the lower the HDLs are. That relationship is extraordinarily strong. So just like HbA1c, that HDL cholesterol is sort of reading out the 24 hour triglyceride metabolism much better than the overnight fasting. Triglyceride measurement is sort of why HbA1c is better than fasting glucose. You know, that's the analogy.

First of all, I've never heard that before and that might be if I learn nothing else on this podcast. Dan, that is hands down the most amazing thing I have learned. Not just today, but I'm going to go out in a limb and say this week, potentially this month. That is super fascinating. First of all, people on this podcast have probably heard me gripe over the lack of integral functions in biology. My background, of course, in math and engineering, we love integrals. Right. As imperfect as the A1C is, as you said, it is an integrator, and it's so hard to find integrators. You know, Ferritin sort of does it a little bit for iron, but not nearly as well. And you know, the holy grail, of course, would be to find an integral of something like MTOR activity or something like that. But I've never before been presented with or confronted with this. So I want to make sure we all understand this a little bit better. So there's a couple things that you've said there. One is if you stick an IV in a person's arm, let's just do that to go easy on them, right? So we're going to just be able to draw blood continuously or call it every five minutes without poking them. You bring them into a lab fasted, you measure their HDL cholesterol, you measure their trig. So let's just say they show up in the morning fasted, their trigs are 100mg per deciliter, HDL is 50mg per deciliter. So most people would look at that and say, oh, that's great. Person looks super healthy. You give them a high fat shake and 30 minutes in, you just start measuring every couple of minutes the concentration of those two things. I think most people somewhat familiar with metabolism would not be surprised to learn that the triglycerides will go through the roof. As an aside, anybody who has accidentally done a blood draw with their doctor after eating a meal when it was supposed to be fasting will be familiar with this.

Right.

We see this from time to time when patients make mistakes. You know, they have a big fat breakfast before blood draw, and you get their trigs back, and they're 4 or 500 milligrams per deciliter.

But highly variable from person to person, just to be clear.

And highly meal dependent as well.

Exactly.

Very much depends on what the meal is. So in this experiment, though, you're specifically giving them something to elicit the biggest triglyceride response. In the example I gave Dan, let's say triglycerides go 100, 125, 150, all the way up to 400 before they start to come down. And that generates an area. You could integrate that on the curve. Can you give realistic ideas or values for what the HDL cholesterol would do during that period of time?

The HDL cholesterol definitely dips during that time in a way that's roughly proportional to the triglyceride levels. But not anywhere near the same degree of magnitude.

Right. Simply because it's starting lower and you have a constraint bottom versus no constraint time starting lower.

But also the dynamics of its turnover are very different. I think the key point here is HDL is not just an integrator sort of acutely over that meal, but chronically. In your example, the HDL cholesterol might go from 40 to 38 or 37, which doesn't sound like a lot after that one meal. But the repeated meals that the individual is eating that are high fat and that repeated triglyceride excursion has a more chronic effect, a little like glucose and HbA1c that keeps taking the HDL down over time until you get to kind of a new steady state. So the acute effect on the HDL is real. It's modest. The chronic effect on the HDL of that abnormal postprandial triglyceride metabolism is quite substantial. And that's why HDL is a good, as you say, integrator of this effect.

And actually, what you just said, Dan, is kind of what I was hoping to go to next, which is, if everything I said were true, that might not be enough to explain. Explain the full integral function, because it probably captures some of what's happening outside of the meal. Just as hemoglobin A1C doesn't only reflect what would be captured in an oral glucose tolerance test. It captures what's happening over 90 straight days when you're eating. When you're not eating.

Exactly. And that's also why there's a very strong statistical relationship between fasting triglycerides and low nihdl. So the fasting triglycerides themselves are still affecting HDL metabolism. It's just that the postprandial part is also a key component.

So let's then again, just one more time, sort of reiterate the lagging nature of HDL cholesterol through HDL biology to what's happening with the efficiency of, maybe, for lack of a better word, lipid partitioning.

The lagging nature, meaning the integrating over time.

Yeah, exactly. Let's use an example. So you've got a person who is exercising and so you have two people who are similar except that one is very insulin resistant and one is quite insulin sensitive. The insulin resistant person is going to have a higher level of insulin. All things equal, they're going to have a more difficult time oxidizing free fatty acid. So as energy demand goes up from the muscle, they're gonna be more likely to utilize glycogen as opposed to utilizing triglyceride. So at any point in time you might measure differences in glucose, differences in lactate, you might. Well, you would see lactate as well, but differences in triglyceride level. You might also notice more fat in the liver or some fat in the liver of the insulin resistant person. None in the insulin sensitive person. In that situation, even when they're not eating, they're quite different physiologically. What is the HDL discerning or doing in those two scenarios that's being reflected in the ongoing integral function of it?

The simplest, most direct model is that the HDL is reflecting the 24 hour excursions and triglycerides like we were just talking about. I think what you may be getting at is there are almost certainly other components of metabolism that the HDL is integrating and reflecting, particularly in the complex insulin resistance world. We know insulin resistance has a lot of effects other than affecting triglycerides. And insulin resistance, I'm sure, has effects that are affecting and are reflected by lowering of hdl. What I can't tell you is exactly what those are. That's an area that is still a topic of, of investigation. I will give you one hypothesis though, and one that maybe I think has some merit. As you know, adiponectin is an adipokine secreted by Fat that has an inverse relationship to insulin resistance. So, you know, basically, people who are insulin resistant have lower levels and people who are, you know, more insulin sensitive. And adiponectin itself appears to have some direct effect on HDL metabolism in the right direction. We could talk about it possibly by tweaking the liver in ways that then the liver affects hdl. So where I'm really going with this, although I have to say the data still are not completely solid, is that another way that insulin resistance is impacting on HDL is through adiponectin secretion affecting HDL metabolism in a way that's completely different than the triglyceride hypothesis that I just put forth. You follow me?

Yeah. And it's funny. We used to measure adiponectin and leptin levels. Again, one of those things I sort of stopped measuring. Do you think that that's a helpful biomarker independently and should that be something that's back on our plate?

Absolutely fascinating. To try to put together these metabolic pathways in terms of its utility as a clinical marker. I guess I'd have to be a little bit skeptical. I'm not quite sure how I would use it in terms of guiding clinical care.

I mean, I think that was sort of where we ended up, which was, look, we get more actionable insight out of other metrics. Okay, that was really interesting. And again, before we leave the pharmaco side of this, can we talk for a moment about niacin?

Oh, sure.

It's been a while since niacin came up on a podcast, but this is an interesting drug because it clearly raises HDL cholesterol and it lowers apob, doesn't it?

It does. It lowers triglycerides, it lowers apob, it lowers ldl. All modestly. But really, Peter, niacin. I just chuckled because niacin is one of these areas where we, as lipidologists and me personally, have to really eat a bit of CRO. There was a time when I prescribed niacin to a lot of patients with primarily the idea that it was the only thing we had to raise hdl. This was in an era where HDL is the good cholesterol and raising it must be good, right? I also told myself, well, niacin does lower triglycerides, it lowers APOB, it lowers LDL. It also, as I'm sure you know, modestly lowers LP as well.

About 15%. Lowering of.

Yeah, 15, 20%. I basically was like, this is a nice, broad spectrum lipid lowering drug that, you know, not in place of but on top of a statin for certain people who had certain lipid profiles, mostly high triglycerides, low hdl, has to be providing some benefit, even in the absence of, as you pointed out, the clinical trials that really are a cornerstone of cardiovascular medicine. Well, the clinical trials got done. The aim high trial, which was a very well done trial, which frankly just didn't show much benefit of niacin, a blow to us. But the field comes up with reasons why that trial may not have been right. Bottom line is one more trial run by Merck with a drug that also helped to potentially address some of the issues with niacin was done very large trial, very well powered trial, and that also had really minimal or very disappointing effects on reducing cardiovascular events. Essentially, those two trials killed niacin. So over the next year, as patients would come back, I would have the discussions that maybe you've had discussions with patients too, like this. You know that niacin I put you on eight years ago that you've been taking religiously, despite the flushing that it causes when you take it? I really think I have to tell you that I'm not sure you need to take it anymore. And it was a humbling experience to basically have a drug that I had prescribed quite a lot, basically to tell patients that. I don't think that in retrospect, this is helping you much. I do have a subset of patients who are so wedded to their niacin that despite that, they haven't wanted to stop. But the vast majority of my patients have stopped their niacin.

And what's the mechanism by which niacin raised HDL cholesterol?

I think one is certainly triglyceride lowering, like we were talking about earlier, but probably not just that, because the increases in HDL were somewhat disproportionate to what you'd predict from the triglyceride lowering. Although again, with the complexity, it's a little hard to make that calculation. So there probably is some other mechanism. And to this day, I don't think we really understand it. We tried for a while to try to figure that out. Niacin is a very complex drug. Of course, we're talking about niacin here in pharmacologic dosing, not in that kind of vitamin dosing. We don't really know. I think it's fair to say how niacin raises HDL beyond its triglyceride lowering effect.

You know, it's not a drug we used much, if at all, but the few times we did, I was actually really amazed at how much it raised HDL cholesterol. I mean, it wasn't uncommon to go from 50 milligrams per deciliter to 90 milligrams per deciliter on a strong dose. Let's shift gears for a second and talk about something that we briefly touched on. But I think we now want to go into a little bit more detail.

Detail.

If everything we've talked about so far is somewhat complicated, I actually think for me, at least, this next part, especially the RCT stuff, gets complicated. So let's talk about what HDL lipidation, delipidation and reverse cholesterol transport are, because this is really where we start to get into some of the sophistication of the HDL and even the interaction with other cells like macrophages and things like that.

I'll start and then you can kind of lead me along because this can get quite complicated.

And maybe just to orient you, Dan, why don't we start with unless you have a better way to do it, I'm totally open. But if you're looking for a goalpost to start in, do you want to start with a foam cell stuck in an artery wall or is there a different place you'd want to start the discussion?

That's a good idea. So many of your listeners know that core concept in atherosclerotic vascular disease is the macrophage that's taken up lipids and is now a so called foam cell, meaning it looks foamy under a microscope because it has all this lipid. And when you stain tissues, the lipids become like bubbles within the cells and they look foamy. The foam cell, the lipid loaded macrophage, is a core pathologic feature of atherosclerotic vascular disease. It's also the first thing you see. So careful studies that have really looked at vascular tissues in children and teenagers and young adults. On the way up, you basically see lipid loaded macrophages accumulating in the subintimal space and the intimal space in the large vessels. Before you start seeing some of the more complex features of infiltration of other leukocytes and extracellular matrix and all the stuff that ultimately comes the complex atherosclerotic plaque. There's been a strong belief for a long time that this is one of the core initiators of the process. And I think that's probably true. An analogy would be like with Alzheimer's, the A beta being kind of the core initiator of the process leading to much more complex pathology. So macrophages have very well established mechanisms for ridding themselves of cholesterol. You know, keep in mind, no cell, except the liver cell, has the ability to metabolize cholesterol to other sterile species. Only the liver can do that. Cells can make plenty of cholesterol, but the only way cells can deal with their cholesterol is to quote, efflux the cholesterol to push the cholesterol out of the cell and get rid of it. So if you think at the whole body level, all of the body is making cholesterol, but that cholesterol ultimately has to come out of those cells into something. This is where HDL comes in and ultimately get back to the liver, where the liver then can metabolize it or directly excrete it into the bile, then it goes out the intestine and the feces. So all cells have to be able to do this, and all cells, in fact, have the ability to push cholesterol out of their cells. But macrophages really have to do it. That is because macrophages are like kind of the dump truck of the body. They're picking up not only LDLs and lipoproteins with cholesterol, they're scavenging cells, dead skulls, apoptotic cells. And of course, all those cells have lots of cholesterol. So macrophages need very, very effective ways to rid themselves of cholesterol. And they do have effective ways of doing that. When those pathways get overcome or less efficient, the macrophage then builds up cholesterol and becomes the kind of foam cell that we're talking about. Macrophages have transporters. They have a very abundant amount of this ABCA one that we talked about earlier. Earlier we talked about it in the gut and the liver. Now I'm saying that macrophages have ABCA1. ABCA1 is one of the not the only way that macrophages rid themselves of cholesterol. If you recall, the main acceptor of cholesterol via ABCA1 Transporting out of a cell is APOA1. This led now quite a long time ago to the general paradigm that macrophage foam cells are building up cholesterol because they're not getting rid of it effectively. And that one of the best ways to get rid of it would be to promote these efflux pathways via ABCA1 and other transporters that are being driven by APOA1 to HDL, which then, by my garbage truck analogy, the garbage truck that is the HDL is picking it up in the periphery, like the blood vessel, and returning it to the liver, dumping it off in the liver, and then going back and doing its job again. This process of, quote, efflux, cholesterol efflux from macrophages and frankly, other cells, particularly in the blood vessel wall, has been for a long time now thought of as a key process that would help to protect against the early initiation and progression of the atherosclerotic plaque. Peter, that was long, but that's a start.

Well, and I think that's. Look, let's just make sure everybody understands what we're talking about here, which is when we really talk about the positive valence of HDL as a particle, it's because of this, right? In large part, this is a big piece of the positive association, presumably, or negative association, depending on the direction of HDLC that we might see, as we've been talking about, really now speaks to function. Part of the problem, it would appear, is that very crude metrics like the amount of cholesterol in an HDL or even the number of HDL particles or the size of an HDL particle, those are basically the only things we can measure clinically at least. Those are so crude and so far removed from providing any quantification of the process you just described that I suspect in part that's why we are stuck in a way that we are not on the APOB side of the ledger, because so much of the damage caused by APOB is simply captured in the number of them, given the stochastic nature with which they enter artery walls and get retained. Would you agree with that assessment?

I would absolutely agree with that. So this process of cholesterol efflux, as these sort of ideas evolved, is, if you will, the first step in this broader physiologic process that Peter briefly referred to that we call reverse cholesterol transportation, or rct, different than a randomized controlled trial. If forward cholesterol transport is basically the cholesterol coming out of the liver into VLDL and LDL and then depositing in tissues like the artery, the reverse transport is the picking up of the cholesterol putatively via APOA1 and HDL and returning it back to the liver, this process of reverse cholesterol transport, plausibly, and I think some data, at least in animal models, is related to protection against atherosclerosis. That is, the more effective you are at the integrated process of not only picking up the cholesterol via efflux, but effectively returning the cholesterol to the liver for excretion, the more you would protect against atherosclerosis, as the theory goes. And I'll just remind you about our example with SRB1. That's the terminal, sort of one of the terminal steps where the HDL is dumping off the cholesterol. If you interrupt that process, your HDL goes up. But the process of reverse cholesterol transport is being constipated, and therefore there's increased risk of atherosclerotic cardiovascular disease. One of the great goals of the field has been, can we promote that first step of the process? Can we figure out a way to promote the driving of the efflux process from the macrophages and maybe other cells in the atherosclerotic plaque to acceptors like HDL in order to protect or maybe even regress atherosclerotic plaque, shrink it as a way of trying to reduce risk? And I'll just say that as we move into this next phase of complexity, I think what's pretty clear is it's not the mature HDL particle. You know, when we measure HDL cholesterol, that's what we're measuring is the cholesterol and the mature particle, that's almost certainly not the particle that is driving this first step of the efflux of the cholesterol from the foam cell and the macrophage. And that's maybe why simplistically raising HDL cholesterol, like with CTP inhibition, doesn't actually reduce atherosclerotic cardiovascular disease events. But are there other ways more creatively that we might be able to drive that process? And also, to Peter's earlier functional point, might it be that different people, even with identical HDL cholesterol levels, have different function of their HDL? One person with an HDL cholesterol of 50 might have something about their HDL pathway that is super functional at driving efflux, whereas another person with the same HDL cholesterol doesn't do nearly as well. And if we could measure function efficiently, might we have a better way of assessing risk and maybe even targeting interesting therapies, More so than just measuring this fairly, not so dynamic measure of HDL cholesterol itself? There's more to say there, Peter, but I'll turn it back over to you.

Well, no, I mean, I think you illustrate the very important distinction between static biomarkers and dynamic biomarkers. And we have very few dynamic biomarkers. You know, in OGTT and oral glucose tolerance, that's the dynamic biomarker in a sense. But most things are very static, and static things allow us to miss flux. So that example you gave of two people whose HDL cholesterol is both 50 milligrams per deciliter, we have no clue what the velocity through the HDL is. In one of those cases, it could be a bump on a log and the HDL is not really doing a heck of a lot. And in the other, that could be the busiest beaver on the face of the earth, just transporting lipid out of foam cells right back to the liver, back and forth, back and forth, back and forth. And that could be the most industrious little guy on the block. And you could be in a totally different risk situation as a result of that. So going back to the rct, how often is it happening that an APOB bite bearing lipoprotein, let's just call it an ldl, is floating around, so not yet in artery wall, but on his way there, presumably, and an HDL come along and they collide in the artery itself and delipidation takes place. So the HDL says, hey, I'm going to take some of your cholesterol away and take it back to the liver now, which, by the way, LDLs do as well. I mean, LDLs are obviously carrying cholesterol.

Back to the liver, but not as a reverse cholesterol transport process.

But yeah, the scenario I described, does that occur often?

No, I don't think so. In fact, as we discussed earlier with ctp, the directional flux of cholesterol in the blood is more from HDL to APOB containing lipoproteins rather than the other way around. So I'm pretty comfortable in saying I don't think that what you just outlined is a way that hdl HDL doesn't directly delipidate ldl. No, no, I think it's more about if it's happening at all. I want to say all this is couched in this is the paradigm that we're dealing with, but where there's still a lot of uncertainty about it. But if it's happening at all, it's really more that something about HDL or something. Apo1 is more interacting with cells to promote efflux of cholesterol rather than with other lipoproteins like APOB containing lipoproteins.

What do you think the future could look like here in terms of commercial assays to measure HDL function? I know that we're a long way away from that, so Maybe I should start with something even more basic, which is, what would it take in the lab to measure HDL functionality if resources were unconstrained?

No, that's where I wanted to go. So, first, I want to say that several years ago, with this concept of reverse cholesterol transport as a dynamic process, we developed an assay in mice that, simply speaking, involved taking cholesterol loaded macrophages with labeled cholesterol, injecting them into the mice, and then following that label all the way through the HDL to the liver, to the gut, to the feces. And we called that integrated reverse cholesterol transport. And we showed in a variety of different genetic and pharmacologic approaches that when you tweak that process either up or down, it mirrored the effect of that process on atherosclerosis. What I'm really trying to say is something that promoted that process and made it more efficient, also reduced atherosclerosis, something that constipated that process, increased atherosclerosis. It gave us a lot of confidence, frankly. It gave the field a lot of confidence. That integrated measure of reverse cholesterol transport is actually relevant to atherosclerotic cardiovascular disease, at least in mice. So that brings me to humans. We can't do the type of experiment I just described in humans. It's not feasible as a research or as a clinical assay. So we thought long and hard about how can we start to think about doing this in people, and developed what I'll call an ex vivo cholesterol efflux assay, where the concept is we took someone's blood or plasma, we isolated the HDL specifically, we got rid of the APOB containing lipoproteins and then put that on cells that were labeled with cholesterol. And we measured the effectiveness by which that HDL removed cholesterol from cells, what kind of cells. And what we found, as I think you know, is we were really struck with how different individuals HDLs were at their ability to extract cholesterol from cells, even with the same HDL cholesterol level. And it sort of affirmed this concept that HDL cholesterol is not really telling us or informing us on the efficiency of the function of hdl, at least in this case, the function defined as the ability to extract cholesterol from cells. And so we went on to use that in larger numbers of individuals and showed that actually that was much more predictive of risk of coronary heart disease than just measuring HDL cholesterol, even when you controlled for HDL cholesterol, statistically and many others have shown the same thing. So I think it's pretty well established at this point that this so called cholesterol efflux capacity measurement of HDL is a better marker or predictor of risk than just measuring HDL cholesterol. Consistent with the idea that function is important and that perhaps if we could increase function, we could maybe have a mechanism for reducing risk. Now, Peter, you asked about the clinical applicability of that assay. You know, I get asked all the time by patients and referring doctors, can you measure my patient's HDL function? There's a lot of effort in this regard. Full disclosure, I was part of starting a little company now several years ago called Vascular Strategies. And Vascular Strategies does do this measurement in a very reproducible sort of way, mostly related to biomedical biopharma research. But there is a lot of interest in trying to bring this clinically. And there are other assays that are being developed. You know, when you're using radioactivity, it's a little, you know, it's cumbersome for a high throughput clinical assay. And others are developing assays that are cleaner and simpler and faster and cheaper. I think there's a chance we could have a more widely available clinical assay to us for this purpose within the next, say, two to three years. Right now it's still under development.

Two follow up questions there, Dan. One is just a technical question on the assay. What type of cell are you using to measure the efflux capacity?

Well, when we first sort of pioneered this work, we used a mouse macrophage cell line called the J774 cell. We have done it with human macrophages as well, and so have others. We like to think that the macrophage is the most relevant cell type for reasons we discussed. Yeah, that's the answer.

And then I guess the second question is using the hypothetical but probably somewhat real example of we'll take 100 people that have an HDL cholesterol that's about the same. We do this assay and we rank order them in effectiveness. You're saying that that rank order of effectiveness correlates directly to risk? Are you saying directly related to risk by proxy, that is other measurements like insulin resistance, apob or are you saying actual outcomes?

The first study we did and published was cross sectional with clinical coronary disease. So basically it was calcium scores or things like that association cross sectionally with prevalent disease. Then we basically said we need to know if this is predictive of incident disease events that occur. So we went to colleagues who did the Eric Norfolk study, which you're probably familiar with, a very large prospective study in the uk, in Europe. They provided samples and we measured this in a very large number, many thousands of samples in a baseline in people who had been followed for 10 plus years. And we showed even in that setting that the efflux capacity was predictive of incident cardiovascular events. So yes, these are hard events, not just proxies like measurement of coronary calcium.

For example, which is again, fascinating. How well does that prediction hold up if you corrected for other things that could be measured in the blood, such as insulin resistance, triglyceride or apob?

We did do the statistical analyses correcting for clinical risk factors, including of course, HDL cholesterol itself. We corrected for things like BMI and presence of diabetes, dichotomous presence of diabetes, if memory serves. We didn't attempt to correct for like a sophisticated marker of insulin resistance like homa or anything, because I'm not sure we even had that data. So your point is well taken. At some point, is this causal or is it still associative, but just even better associative because it correlates with things even better than HDL cholesterol. And I think that's why we need, of course, interventional studies that actually test the hypothesis.

Well, or even could we use it? There could be a hybrid there. Right. Which is it's absolutely causal. But because it's so difficult and complex to initiate at scale, what if we use AI to figure out that it's equivalent to a new metric that is a composite metric of things that we can measure as biomarkers, if that makes sense.

I hear what you're saying, and that would be a great thing to think about. In our paper in Epic Norfolk, we did correlations of the efflux capacity with lots of different things. I frankly have to go back and look at that and remind myself if we had like fasting insulin or anything, because I think that starts to get more at the insulin resistance component. But you're absolutely right. We may be able to find markers that in composite would predict efflux capacity rather than having to measure the efflux capacity itself. I'm not convinced we'll be able to do that, but I think that it's.

A good thought because the analogy that comes to mind is, you know, certain labs use a series of NMR metrics to predict insulin resistance, which I've always found not that interesting, frankly. Cause we can measure insulin resistance so easily that using NMR to add another insulin resistance metric.

I hear you.

It seems a bit backwards, but the idea was they were able to basically look at the NMR spectra of all of the lipoproteins and impute a composite metric that came back. So if you could do that in the other direction, which would actually be the valuable direction, it could be amazing. In other words, if there was an HDL function score built out of X, Y and Z. Anything else? Before we leave the topic of kind of measurement, we haven't really talked about the NMR side of the equation. That's probably a bias because we don't use nmr, but clearly there are labs out there that will count the number of HDL particles and spit out an hdlp. Anything you want to say about HDLP or anything like that?

Yeah. The overall number of particles is obviously correlated with HDL cholesterol, but is a different measure because HDL cholesterol is just that amount of cholesterol carried in the hdl, whereas the particles are more analogous to sort of measuring apob, meaning total particle number. I think the data on balance suggests that HDL particle number is a little bit better than HDL cholesterol at predicting risk. We haven't formally compared HDL particle to cholesterol efflux capacity, the functional measurement. Fundamentally, it's another static measure though. And so while it has maybe some limited predictive value, I don't think it takes us that much further in terms of trying to get at where we're really trying to go is better predictions of risk, particularly in a setting where we might want to intervene to actually try to reduce risk.

Let's talk about something you mentioned kind of in passing a couple of times.

Peter, before we leave this topic though, I do think it's important to remind your listeners that this concept of promoting the efflux and reverse cholesterol transport pathway is being tested with another intervention called CSL112. It's a form of APOA one that's been complexed with lipids in a so called recombinant HDL particle is being tested for impact on cardiovascular outcomes in the setting of acute coronary syndromes. So people have come in, been randomized to four weekly injections of this APOA one containing recombinant particle, which is very effective at promoting cholesterol efflux based on all the work that's been done, with the idea being that this may directly impact the plaque and impact on cardiovascular events. So this is I would say the closest thing we have to a formal test of the cholesterol efflux hypothesis as an intervention in terms of whether it will reduce risk. Those of us in the field are on the edges of our seats. You know, it'll be a little while to really see what this trial shows. I do think it's going to be interesting either way.

So earlier, Dan, you mentioned kind of briefly the association of plasma APOA1 and HDL cholesterol with neurodegenerative diseases such as dementia or Alzheimer's disease. I don't know if it extends to Parkinson's or Lewy Body. What do we know about that?

Well, this is, I think, a fascinating new area of HDL biology that has a number of components in that that's still being investigated. I want to maybe start by bringing in apoe. We haven't talked about apoe. APOE clearly has a role in apob containing lipoprotein metabolism and mediating the uptake of those remnant particles into the liver.

Can you actually explain this a bit more? Because most people hearing this are going to think of apoe. The gene, and of course the gene for APOE people are going to be familiar with it exists in three isoforms, 2, 3 and 4. But of course these things combine. So you have six combinations of an APOE genotype and of course the gene codes for a protein. So I mean, I assume everybody has the same APOE protein. You're going to have different amounts of it and different, potentially functionality depending on the combination of which gene produced it.

Exactly. APOE is possibly one of the most fascinating genes and proteins in human biology in terms of its roles and its, and the isoform issue and its relationship to disease. So as you point out, there are three common isoforms of ApoE. ApoE 3, being the so called wild type, are the most common. You know, in lipidology we've focused a lot on APO E2 because if you inherited two copies of APOE 2, that form of APOE is defective in binding to the LDL receptor and the other receptors that mediate uptake of remnant lipoproteins, both chylomicron remnants and VLDL remnants. And therefore if you homozygous for ApoE2, you're at risk for so called, again the terminology is bad type 3 hyperlipidemia, which is basically a remnant clearance disorder. These individuals can't clear their remnants appropriately. They get high triglycerides and cholesterol. They also are at increased risk of atherosclerotic Cardiovascular disease. It's a classic lipidology thing.

And these patients require fenofibrates to bring down the VLDL and triglyceride.

They respond to the a lot of the standard LDL lowering therapies like statin and ezetimibe. They Even respond to PCSK9 inhibition. But sometimes we have to add on fibrates as well to maximally control them. Depends on how severe they are.

And yet those people. And by the way, that's the only genotype I've never seen. It's much more rare than the 4:4 homozygotes. But the tutu would come with about a 20% risk reduction, relative risk reduction in Alzheimer's disease, but paradoxically comes at this higher risk of ascvd.

Exactly. So comes at higher risk of lipid disorder and ascvd. But that's exactly why I brought up APOE in response to your comment about neurodegenerative disease. So as probably a lot of people know who are listening, the APOE 4, as you just pointed out, the APOE 4 form, which is present in about 25% of people, so it's quite common. And even in the homozygous form, certainly plenty of people out there who have both two copies, is in a dose dependent way the major genetic risk factor for Alzheimer's disease. No question about it. And as you said, Peter, Apoe 2, which is bad for your lipids in the blood, is actually protective against Alzheimer's. It's absolutely fascinating biologically. There has been so much work to try to understand how APOE is interfacing with Alzheimer's disease. So APOE is made in the brain. It's made by cell types like microglia in the brain, and it's a lipid binding protein that transports lipids. And now we know from genetics of Alzheimer's that there are lots of other lipid genes that are related to Alzheimer's. So the story starts with the fact that APOE made in the brain is somehow impacting on Alzheimer's risk. And if you have this form of APOE4, and especially if you have two forms of APOE4, you were at very substantially increased risk of Alzheimer's for reasons that we still don't fully understand. But as a lipidologist, I think it has something to do with lipid transport in the brain. So that brings me to hdl. The APOE in the CSF and in the brain is mostly made there. The newer developments that are relevant to APOA1 and HDL are that there is clearly APOA1 in the brain and in the CSF. APOA1 is not made in the brain. So APOA1 gets there somehow through the blood, which I'll come back to. But there's now quite a lot of observational data that strongly suggests that APOA1 is protective against neurodegenerative disease. Now, this is associative data. It doesn't prove causality. But the reason I think it's plausible is that two of the major genetic risk factors for Alzheimer's that are expressed in the brain are ABCA1. Remember that lipid transporter that rid cells of cholesterol and that Apoa1 interacts with and promotes? And Abca7, a very close relative of Abca1 that structurally looks very similar and that we don't know exactly what it does. But I think it's a safe bet that it's transporting some sort of lipid to something in the extracellular space, whether that's Apoa1 or Apoe or something else. So the plausibility of Apoa1 being protective against neurodegenerative disease, particularly Alzheimer's, is quite high. And frankly, there's a lot of work to be done to try to figure it out. There's one other point I'll make, which is the blood levels of Apo1 and the CSF levels of Apo1 and the relatively small number of studies that have measured both in the same people are not that well correlated. So it's not simply a matter of if you have a high level of APOA one in the blood, that's going to generate a high level of APO1 in the CSF. So what that probably means is the processes that are happening that get the APOA one across the blood brain barrier are highly regulated processes that, a little like the cholesterol efflux story, probably differ from person to person and aren't directly being driven by the level of APO1 in the blood. Where I'm really going with this is this concept that if we could figure out how that happened, if we could Somehow promote more APO1 going into the brain, into the CSF, the data suggests that that might be a very interesting opportunity to blunt or reduce risk of Alzheimer's. I find it fascinating, as you can tell, Dan.

Do we know if this relationship is stronger or weaker as a function of APOE genotype? Is the effect more pronounced? Do we have enough data to parse out that type of a relationship? So I'll give you an example.

Yeah, that's a really super Question. And I don't think we have big enough data sets yet to be able to parse it out in terms of by APOE genotype to figure that out. But I have to go back and look for that. But I don't think we know that yet.

Do you have any idea why labs have not developed what would be very easy to do, which is just a commercial assay for APOE concentration? There's certainly some data to suggest that APOE concentration might be more relevant than APOE genotype, although of course it's highly influenced by APOE genotype. But you could almost think of it as a gene expression measurement. Accrued measurement of gene expression for apoe. Is that something that you've seen or used even in the lab?

You're talking about APOE in the blood, right? Not in the csf? Just to be clear, there are automated assays for apoe. We run one routinely in our lab. I think it's not a clinically used assay.

No, I've never even seen a CLIA based assay for it.

Right. I think mostly because it isn't that helpful in the limited studies that have been done, which admittedly maybe aren't as voluminous as they should be. It isn't that really helpful in terms of predicting cardiovascular risk. Total plasma apoe.

Oh, I was thinking more for neurodegenerative disease. Has that been looked at?

Oh, well, you know, I didn't say it, but the levels of APOE in the CSF and the levels of APOE in the blood for the few studies that have measured both in the same people also do not correlate much at all.

You would need CSF APOE if you wanted to do anything with this.

Exactly. That's what I'm getting at. You would need to be able to do it. I would not be surprised if someone develops an assay for APOE concentration in the CSF as a clinical tool, because there I think that might be highly relevant.

Interesting. So here we have basically HDL potentially being protective in the brain and presumably doing so via Apoa1, potentially offsetting some of the APOE problem. APOE being as I think one of our previous guests referred to as kind of the general contractor of cholesterol in the brain.

Yeah.

Does HDL do anything with nitric oxide? I think I remember reading something about promoting sort of endothelial NOS activity, nitric oxide synthase activity. Is that a major issue?

There's been a variety of other functions that HDL has been reported to do, which I think Are absolutely real. Their relevance to human disease and pathophysiology, I think, are less clear. But one example is what you just said. So a couple investigators, most notably Phil Scholl in Dallas, has shown very clearly and beautifully in cells and mice that HDL can promote nitric oxide production in a way that would be expected to be beneficial both in terms of blood pressure lowering and maybe protection against atherosclerosis. And that SRB1, the receptor we've been talking about, is also present on endothelial cells and mediates at least part of that effect of hdl. It's a fascinating observation that is absolutely, I think, solid. Translating that observation to relevance in humans, I think is challenging, but I think plausible. Another, we've been talking about insulin resistance. I'm sure you're aware of the data that suggests that HDL can interact directly with skeletal muscle in a way that promotes insulin sensitivity.

No, I'm not.

Fascinating work. Again, I put it in the same category of solid. In terms of what it's been able to show, mechanistically, a little bit unclear and relevance to human disease and physiology, I think still a little bit unclear. These are just two of the other types of things that HDL has been shown to do that would be putatively beneficial but are of uncertain relevance to real human disease.

You know, it's even more sort of removed than that, because even if we knew mechanistically that this were sound, we're still back in the same area we are with ascvd. In ascvd, there's really very little ambiguity about the utility of hdl, the beneficial utility of hdl, and yet we're standing here with our hands in front of us saying, well, what can we do about it clinically? As a physician, as a patient, what do I get to do about this knowledge that HDLs are helpful particles when I can't measure the manner in which they do their job? And the only things that I can measure are as useless to me as my eye color, basically. And then we're expanding into. And look what they can do in muscles, and look what they can do in insulin sensitivity, and look what they can potentially do in the brain. It really comes back full circle to how we open the discussion, Dan, which is this is such a complicated area of biology that I would guess it has to be considered the next frontier of the lipid space. I mean, when we take a step back and put ASCVD in the context of cancer and neurodegenerative diseases. Right? So these are the Big three killers. In the modern world, we know so much more about ascvd and we have so many more tools to effectively treat it. We know a bit about these other diseases, but in the case of Alzheimer's disease, we don't have a single tool to do anything about it. In the case of cancer, most of our tools do nothing. 90% of the tools do nothing. That is, they barely extend median survival but don't cure people. But in ascvd, we can really move the needle. And yet, you could argue, half of the field we still know nothing about. And is there going to be a renaissance, you think, or are we up against some technical limitation on this inability to measure function? And I say that in a practical way. I mean, yes, I think you're already measuring function in the lab, but is this a bridge too far for the clinician?

Well, I'm going to separate that into using a measurement of function for risk prediction and then the implications for intervening therapeutically. I think that HDL function, say cholesterol efflux, has the ability to allow us to more specifically assign risk better than just measuring HDL cholesterol. I think realistically we need a good, reproducible, easy to run, automatable assay that then is tested in large numbers of people and shown to predict risk better than HDL cholesterol itself. I feel like knowing what I know is going on in terms of developing those assays that there's a decent chance that we're going to get there. But it's not just the development of the assay, which actually there are several assays now that are. But it's the proving that the measurement actually enhances risk prediction enough, not just incrementally, but enough to make it actually worth doing in clinical practice. I think there's a. I'm not going to say 100%, but I think there's a more than 50% chance that we're going to see at least some assay come out that will allow us to do that. And then it will be, frankly, physicians like yourself and like Tom Dayspring, who pick up that assay and start using it in their patients and start trying to get a sense for whether it's useful in the context of a sophisticated preventive clinical practice. I think with regard to intervention, the study I talked about is huge in terms of this. Let's just assume that that study is positive. Four infusions, weekly infusions, significantly reduces cardiovascular events in patients with ACS after 90 days. If that study is positive, if you think about that, the implications for that product, but more importantly for the field that showing that actually triggering cholesterol efflux via infusion of that particle, that type of particle that will rejuvenate the field of cholesterol efflux and reverse cholesterol transport as a therapeutic target for intervention. I think if that trial is not positive, I think the concept of intervening around HDL and reverse cholesterol transport for purposes of ASCVD is probably past recovery. That would be my view. And even that CTP inhibitor that I mentioned that's still in development, it's really more about reducing APOB than it is about raising htl.

When does that trial read out? The function trial?

I should have refreshed my memory on that. I just can't remember.

Where is it being done? How many centers roughly?

It's a global study. There's centers in US and Australia and Europe. It's a classic global large cardiovascular outcome trial. I think the leaders of the trial I think are in Boston.

Remind me, does it have a fancy acronym yet?

Yes, and I'm embarrassed to say that I'm not quite coming up with that either.

We will figure that out and it'll be in the show.

Notes if I could just follow up on your next frontier comment, I don't think HCL per se is the next frontier, but I do think lipid metabolism in the brain is one of the next frontiers. I really do. With regard to neurodegenerative disease but also other brain function. I say that as a long term lipidologist who has focused primarily on the blood. So I admit my biases and looking for things that we can apply as lipidologists our expertise to. But I do think understanding lipid metabolism in the brain and its relationship to disease, you know, the brain is of course the most lipid rich organ of any organ in the body has been an under investigated area that we have a lot to do. But I think we're going to be uncovering some very interesting things that with luck have implications for therapeutic intervention to prevent neurodegenerative disease.

Dan, it's hard to go anywhere from here that is, I mean both exciting and important in ways that are rarely captured even in this subject matter. So I want to thank you for taking a subject matter that is so complicated that I've largely shied away from really doing anything on it in the AMA series that we do and so eloquently describing it and really using. If someone's listening to this and this is the first podcast they've ever listened to of ours, they might think, oh my God, that podcast is too technical. But I think for regular listeners they will appreciate that you really did a great job simplifying a very complicated subject matter and certainly did a better job than I would have ever been able to do. Nothing simply because I don't know as much, but because the ease with which you were able to speak about this is impressive. So thank you for taking the time. Thank you for doing that. And most importantly, of course, thank you for your work in this field. We, I think, collectively look forward to seeing how things shake out over the next decade.

Thanks very much Peter. I really enjoyed it.

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