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Welcome to a special episode of the Drive. For this week's episodes, we're gonna rebroadcast my conversation with Gerald Shulman, which was originally released, I think, December of twenty twenty. This episode is really a masterclass on insulin resistance. In this, Jerry clarifies what insulin resistance means as it relates to muscle and liver and the evolutionary reason for why it exists. He goes into great depth on the mechanisms that lead to and resolve insulin resistance.
the clinical implications of these mechanisms, the role of diet, exercise, and pharmacologic agents.
Well this was one of our most technical episodes. It was also one of the most popular episodes ever. Unfortunately, the complexity of this episode is the price one has to pay if you really want to understand longevity. You're gonna have to understand insulin resistance. So To help with this topic better, we then released an AMA in February of twenty twenty one, I believe it was AMA number twenty, called Simplifying the Complexities of Insulin Resistance.
And in that AMA, I basically sat down with Bob Kaplan and we went through the podcast and tried to explain some of the more complicated areas in it. This is another great resource for people who want to go deeper into this subject matter. So Just as a brief reminder, Gerald is a professor of medicine, cellular and molecular physiology and the co-director of the Diabetes Research Center at Yale. In twenty eighteen, he received the Banting Medal for Scientific Achievement.
Which is generally regarded as the most prestigious award one can win in this field. So without further delay, please enjoy or re enjoy my conversation with Gerald Schulman. Jerry, thank you so much for making time to sit down virtually with me today. As I said before we hopped on, this is a topic that is near and dear to my heart, and frankly
All roads seem to point to you. And that goes back to, I don't know, at least for me, probably 2011, when I became really fascinated by this topic. And there aren't a lot of topics. Where I've personally experienced the following problem, which is the more I think I understand it, the less I do. So now when someone says to me, Peter, what's insulin resistance?
You know, I can sort of give glib answers to that question, but the reality of it is I don't think I fully understand what it is. And I don't know that I can represent to the listener that by the end of this they will fully understand what insulin resistance is. But what I think they'll understand is how maybe we can think about it through the lens of different tissues and what may or may not be going on. And in large part, I think that's due to the incredible work you've done over your
entire career. I I guess I'd like to kinda just start with a little bit of background. You did an M D and a PhD and you're trained as an endocrinologist, correct? Yeah, that's correct. And then I did residency in medicine at Duke, uh, fellowship in endocrinology at the Mass General, Harvard, and then I've always been interested in metabolism, diabetes. I guess probably my father was a diabetologist, went to summer camp. He was the doctor for type one diabetic. at an early age is exposed to
problems type one diabetes in my peers. I was just a camper and saw my peers getting hypoglycemic or getting into issues with ketoacidosis. So I think I was exposed to metabolism at an early age. I'm sure it left an impression on me. My father wanted me to become a radiologist'cause of my physics background, but I ended up staying in metabolism and and doing endocrinology.
I'm sure you would have done great things in radiology, but I also think we're far better off for the contributions you've made in this field. When did this particular question of understanding what insulin resistance meant and actually starting to differentiate between some of these phenotypes of
What is the fate of glucose in a person with normal metabolism versus what is the fate of ingested glucose in someone with type 2 diabetes? When did that question begin to obsess you? And specifically, now that's a sort of change from the patients that you grew up with with type 1 diabetes.
Studying in as an undergraduate in medical school, I was always interested in biochemistry, physiology. I had an experience. I was visiting a medical student at Vanderbilt in the seventies and got interested in in vivo metabolism, doing studying metabolism in awake animals, looking specifically at glucose and fatty acid turnover, using tracers to actually measure how fast things are being made, glucose is made, how fast fatty acids are being made in the body and metabolized.
in medical training you go back to medical school, you you learn how to become a good doctor, take care of patients. But then in your fellowship years, you're back in the lab. And I really wanted to get back to understand metabolism by looking inside the cell. So everything I had done prior to then and most people studying biochemistry, physiology would to understand so diabetes, metabolic disease I was
interested in this question. It's an important disease, leading cause of blindness, end stage, renal disease, leading cause of non traumatic loss of limb cost to US society is huge impact and now it's becoming a global problem as they adapt to westernized diets and things.
And I wanted to look inside the cell, metabolism inside the cell. And so that took me into the world of nuclear magnetic resonance spectroscopy and actually brought me down to New Haven where they were s just setting up methods, this technique to actually look inside. living yeast cells. I said, gosh, this we can adapt this to humans and look inside metabolism in humans, in liver, and muscle and on other organs.
To specifically get at your question, I think it's such an important metabolic disease, the most common metabolic disease. And so someone who's interested in metabolism, it's a natural segue. I sometimes describe it to patients as the foundation upon which the major three chronic diseases sit. So You described some ways in which patients with type two diabetes die, specifically through amputations or complications of amputations, such as infections, and obviously through end stage renal disease.
But I would argue that the majority of the mortality through diabetes comes not so much through diabetes, but through its amplification of atherosclerotic disease. cancer and dementia, all of which are force multiplied in spades by type two diabetes. So the way I explain it to people, and I hope that by the end of this you'll help me refine this, because it may not be accurate, but I describe to patients that there is a continuum from hyperinsulinemia to impaired glucose disposal.
To NAFLD and NASH to type 2 diabetes. And that continuum makes up a plane upon which all chronic disease get worse. If we're going to be serious about the business of delaying the onset of death, we have to be serious about the business of delaying the onset of chronic disease. And if we want to do that, we must fix our metabolisms. That's my thesis.
Total agreement. You're spot on. So insulin resistance is the main factor which leads to type two diabetes, but it also and again this is give credit to Jerry Revan who in his nineteen eighty eight Banteen lecture first got everyone's interest in basically saying insulin resistance is not only leading to diabetes, but as you say, atherosclerosis, basically hyperlipidemia. Associated with inflammation, high uric acid, polycystic ovarian disease.
Now we can kind of add to that. Now we can talk about NAFL or I prefer the term metabolic associated with fatty liver disease, MAFHL D. That's the going to be the most common cause of liver disease, liver inflammation, end stage liver disease, and liver cancer. And finally, another arm, you know, for Jerry's circle of insulin resistance and all these arms butting off of them, heart disease, as we talked about, high uric acid, high triglycerides and high cholesterol, is cancer.
So we're now, as you know, seeing huge increases in many forms of cancer s which are associated with obesity, breast cancer, colon cancer, pancreatic cancer, liver cancer. And by debt, it's insulin resistance that's driving the increase in all of these cancers. Now it's not causing them necessarily, but it's promoting the growth. And again, we have very strong preclinical evidence for this in animals. You can take
animals. Rachel Perry, who was in my group and now starting her own lab, has taken breast cancer models, human breast cancer models, colon cancer, put them into mice and just giving them insulin, putting an insulin pump, just simply and that the rate of tumor growth is accelerated and you treat them with an insulin sensitizing agents and you can slow down tumor growth. So I think you're spot on, Peter. Insulin resistance
is driving a lot of disease. And you're also spot on in that that's what's killing our patients with type two diabetes. It is heart disease. These other things are the chronic complications of hyperglycemia, the blindness, The end-stage renal disease and the small vessel disease leading to non-traumatic loss of limb. Also hyperglycemia, but insulin resistance. which is very common. It's probably one quarter of our population, one half of our population has it perfectly asymptomatic.
you don't know you have it. We can test for it using sophisticated tools that we can talk about, but it's a very common phenomenon. So before we launch into what I think is an important discussion around the fate of glucose under normal conditions, which is the backdrop against which I think everything we are going to talk about has to be laid out.
I'd like you to spend a moment doing something you're probably not asked to do often, which is at least explain to some extent what the NMR technique allows you to do. Because so much of what we're going to talk about today requires either a leap of faith that you know what you're talking about, or at least some sense of how a scientist is able to actually look at substrates and substrate utilization and substrate movement.
in the ways that we have to be able to talk about them at a molecular and cellular level to make sense. So I know it's a bit complicated, but because it is such a cornerstone of your work. Can you explain what labeling means and how you can measure those labeled molecules in vivo? In metabolism, the traditional methods since going back to dates maybe uh fifty years ago, when you wanted to measure
More than just concentration of a metabolite. You go to your doctor, you measure blood sugar, cholesterol, and it's a static concentration. And what we know is what's much more important than just measuring concentration is flux. And that's basically production versus uptake by a tissue and know where something's being made, where it's going. And the traditional approach has been to
put a label on that whatever you're interested in tracing, uh glucose. And so you used to basically with the advent of cyclotrons it really started in California, in Berkeley, they started, you know, had cyclotrons are interested in nuclear theory, the side product is you can make isotopes. So you can make carbon radio labeled, so it's an emitter, and put that carbon onto a glucose molecule and then trace it. So For more than fifty years we've been able to buy radio labeled isotopes.
And put a carb in, C14, which is radioactive, low-dose radiation, or tritium, which is a form of hydrogen, and then give it to a person, animal, and do blood sampling and actually measure then turnover of that metabolite. So that's telling that's very important information. Many, many important studies have used this, and to date we still use this, to track production and clearance of whatever we're labeling. What you can't get from that though is really what's happening inside.
the cell, which is really where I wanted to go. So we've been measuring turnover of metabolites. Again, that's what I did many years ago where I first started my interest in metabolism.
To do that, you need to get inside and look at the cells. So the approaches have been traditionally something called positron emission tomography, which is now used clinically sometimes to track tumor growth because tumors take up glucose and you can give a pet emitter of glucose and then see if the tumor's taking it up.
That's radioactive and again I'm a clinical physiologist. I'd prefer not to give radio labeled substrates, radioactive substrates to volunteers who volunteer for my study. The other approach was nuclear magnetic resonance spectroscopy. There, you know, were two groups that were pioneering this kind of work. There was one group in George Rada at Oxford, and this was phosphorus and amar. And so what George was doing So NMR takes advantage of the fact that the nuclei
of certain atoms have spin properties and I won't get into all the physics behind this, but they make them behave like tiny bar magnets. And so when you put them in a strong magnetic field they tend to line up or against this magnetic field. And because they have spin properties, they will actually precess in this magnetic field at a set frequency.
if you pulse them at the frequency that they're precessing, they tip out of this field and then when they relax they emit energy that you can pick up with a little radio with an antenna and basically get chemical information about where that label is within a molecule. So Everything I just said, all you need to understand is you can use this method to basically measure the amount of the metabolite. More importantly, which for example carbon
atom within that glucose molecule is labeled. It has something called chemical shift. It experiences a slightly different magnetic field depending where it is within that glucose molecule. So for the listeners, all you need to understand is using this method we're able to get biochemical information of not only measuring a metabolite, but then using the power of, for example, C thirteen NMR.
Track the label as it's being metabolized inside the cell. So that's carbon NMR. So in our bodies, 99% of the carbon in our body is. C twelve, which is NMR invisible, but 1% is C thirteen, which is NMR visible, has this precession properties. You can use a labeled, for example, C one labeled glucose and then track that C one glucose as it gets into the say a muscle cell or liver cell and gets metabolized and finds its way into glycogen.
And then you can measure flux. You can actually, for the first time in humans non-invasively, without any ionizing radiation, measure how much is going in through measure intracellular pathway flux. Phosphorus enamar, as getting back to George Rod, George pioneered phosphorus enamar. There you don't have to give any isotopes. you actually see P thirty one, phosphorus thirty one is a hundred percent natural abundant. You see all the phosphorus that's in solution in our bodies. So
For example, when our volunteers go inside a magnet and we put a leg or arm into the magnet, we can see all the high energy phosphates in, for example, ATP. Edenosine triphosphate. There are three phosphates. And you can actually see each one of those phosphates. You can see phosphocreatin has a different chemical shift. You can see inorganic phosphate.
And we developed methods, Doug Rothman and and others at Yale who I worked with were able to develop methods to measure glucose six phosphate. So we can actually look at one a key intermediate getting glucose from outside inside.
Another method we developed was we can measure intracellular glucose inside human muscle non invasively. So by measuring these metabolites, measuring flux, we can actually then ask the very simple questions, which this is how we started out in humans, as you say, you know, again, diabetes.
is a abnormality of metabolism. Glucose is the metabolite. And we were able to basically ask very simple questions when a person, a human, which is my favorite model because it's the one most relevant to understanding diabetes and metabolic disease. When we ingest carbohydrates. How much of that carbohydrate ends up in glycogen versus oxidation into carbon dioxide or gets converted to lactate through glycolysis? And so and then more importantly in the patient with
Or the volunteer with diabetes, how important is that pathway, glucose to glycogen, accounting for their insulin resistance? The story is very short. But before you go there, let's demonstrate clinically a difference between these two people. So let's take the normal patient. without type two diabetes and then let's contrast them with a very similar person of similar size who has type two diabetes. We will feed them both a high carbohydrate meal in the evening.
Let's just assume that that meal contains one hundred to two hundred grams of carbohydrate. They will digest their food. We won't really have much insight into what's happening overnight. You will tell us. But at the next morning, we do a fasting blood glucose level on them. This is now 12 hours after their meal.
The patient who does not have type two diabetes might arrive with a blood sugar of one hundred milligrams per deciliter, which we will say is normal, His counterpart with type two diabetes may actually at that time have a blood sugar of two hundred milligrams per deciliter, which is obviously abnormal and consistent with the diagnosis of type two diabetes.
Now of course, that only represents about an extra five grams of glucose in the circulation, that is the difference between the one hundred and the two hundred milligrams per deciliter. which is a small fraction of the call it one to two hundred milligrams, pardon me, grams rather, five gram difference. So it's a small fraction of what was ingested the night before. What is the difference between those two people? Why does one of them have such a hard time with that extra five grams of glucose?
What was the fate of glucose in the healthy person to begin with? How did the body treat it? The body when you take in and again this is what we were able to demonstrate by actually measuring glycogen flux in liver and muscle. That ingested in a healthy individual ends up as mostly liver and muscle glycogen.
It takes up muscle and and depends on the size of the meal and how it's being administered, the proportionality between liver and muscle. But the bottom line, it's eighty, ninety percent is stored as glycogen. In the diabetic contrast is there's two processes that have gone awry. One is that the liver is geared up to make more glucose. than it should be through a process called gluconogenesis, the conversion of non-glucose precursors like amino acids, alanine, and lactate to glucose.
And that process is accelerated. So the liver's making twice the amount of glucose as it should. And then you have a block in the periphery where the glucose, same amount of insulin is not causing the glucose to be taken up by the muscle. Again, in the in terms of flux, what I care most about production is up and clearance or disappearance is down. And besides this, also, even in some diabetics, insulin's inappropriately low because we know if we give more insulin
we can overcome these abnormalities. And so the beta cell has also become abnormal in the established diabetic where it's not making enough insulin. that can be secondary to these other issues, glucose toxicity and other factors that have caused this beta cell impairment, because we know Most importantly, when we reverse the insulin resistance, this is a very important study, is we've taken these type two diabetics and short term hypocaloric feeding, twelve hundred calories a day,
we basically can reverse all these abnormalities through reduction in a topic lipid, which we can get into molecular mechanisms and reverse their diabetes. And this has now been shown
by many, many other investigators and most recently Roy Taylor, my colleague who trained with us, is now doing this in the primary care clinic uh back in the UK. But usually you've asked the question, usually when we talk about Diabetes actually it's it may be easier to understand when you start in the young, lean twenty year old. who already has instant resistance. These are the young, lean,
college students that we study. It's actually easier, I think, for your listeners to understand if we start with just pure insulin resistance, which we see as the most common thing, as I said, Probably half the people in the US actually have insulin resistance don't know it because they're asymptomatic. And we even see this in young, lean 20-year-olds, Yale undergraduates who volunteer for our studies, profound insulin resistance in muscle, no problems in liver.
And then take you through the progression from how you just go from insulin resistance in muscle to fatty liver and insulin resistance in the liver and then progress to type two diabetes. That's something we can actually go through if that would be of interest.
It would because it actually kind of fits with the way I was going to try to temporally split this, which would look as follows. When we take a patient who has normal Fasting glucose and normal fasting insulin are And we challenge them with an oral glycemic load and then measure insulin and glucose in thirty minute intervals A lot of times we expose a problem that seems most easily explained by the muscle's inability to assimilate glycogen.
So a person shows up and they have a normal fasting insulin, say it's five, and their fasting glucose is say ninety. You challenge them with 75 to 100 grams of glucose. But say thirty or sixty minutes later, their fasting glucose is two hundred, their insulin is seventy, we call that insulin resistance. And we impute from that that something has broken down in the pathway that prevents their muscle from taking in glucose. Now you've done very elegant work to examine
all of the possible places that failure could have taken place. Did it take place At the GLUT4 transporter, or one of the mechanisms, which we should discuss how the GLUT4 transporter gets across the cell membrane. Is it a problem not of br bringing glucose in, but really is the problem downstream at hexokinase or glycogen synthase, things like that. So
Is that sort of what you're saying, which is can we start with postprandial hyperglycemia? Yeah, I think we're not even hyperglycemia. This before any abnormality just insulin resistance. What I like about the question you asked and how you pointed out, insulin resistance is the root cause for not only diabetes, but it's gonna be the root cause for all these other abnormalities, fatty liver disease. Make us prone, makes a lot of cancers worse.
Heart disease, and again that's the number one killer in this country. It's insulin resistance that's driving all these things. And not even talking about even though I'm a diabetologist, I of course care I want to fix diabetes. But even before blood sugar goes up, which is how we define diabetes, let's understand insulin resistance, because if we can understand insulin resistance, then that's going to be the best way to fix diabetes, type 2 diabetes.
Heart disease are gonna make a big impact there, fatty liver disease and slow down cancer. So let's start with insulin resistance. Okay. What is insulin resistance? So we define it by giving insulin and we know insulin normally does some effects makes glucose being taken up by liver. and muscle and when that same amount of insulin is not doing these things, we say it there's insulin resistance. So you need more insulin than to cause muscle to take up glucose or the liver to turn off.
glucose production or take up glucose. And the same thing again in the fat cell. What insulin does in the fat cell is the puts the break on, break down of fat, we call that lipolysis or take up glucose to esterify fatty acids into glucose. So these are the three key insulin responsive organs, and when insulin is not doing that properly, we call that insulin resistance. And again,
I keep emphasizing I think for your listeners, this is probably every other person in this country and uh in Western Europe are insulin resistant. Your doctor won't even know this unless they do careful maybe studies to assess insulin resistance. Because you won't pick this up as with a simple plasma glucose test. So what causes resistance? Let's start with muscle.
And the reason I like to start with muscle is when we study our young volunteers, again, I like them because they're perfectly healthy. They're twenty years of age, nineteen to twenty. They're lean. Because we know everyone who's overweight or obese probably has insulin resistance. There's so many confounding factors that happen in overweight obesity. These are lean twenty-two, twenty-three BMI.
lean. Non-smoking, so we screen out smoking. No medications, no drugs. And sedentary, because we know people who exercise, we can reverse insulin resistance and we can talk about how that happens. So you give these young twenty year olds, let's say you screen we screen to this date probably a thousand, but you get a distribution, given a drink of glucose tolerance, 75 grams.
You measure insulin and you can calculate insulin sensitivity index. It's a crude index, and it's kind of a bell-shaped curve. And you have people in the bottom quartile who are insulin resistant, by definition, in the top quartile. Then you ask why are those people in the bottom quartile insulin resistant? And you measure glycogen synthesis using the methods we talked about briefly, carbon NMR, give C1 glucose, measure flux into glycogen.
And it's already down by 50% compared to the sensitive ones under matched insulin and glucose concentrations. So they're resistant. Because they can't get glucose and glycogen. That's the major pathway. It's not glucose to lactate, not glucose oxidation. So that's your pathway. Then you want to know where the block in that pathway is.
With phosphorus NMR we can measure glucose 6 phosphate inside the cell. With a carbon NMR method we can measure glucose inside the muscle cell. The reason this is important is we can See where the biochemical block is. So your listeners all sh probably you know, get into a car and they're on the road and if there's construction going on, we all know construction piles up after that whatever that roadblock is where the construction's happening. Biochemistry is the same thing. You have a roadblock
and traffic builds up behind it. So we measure G6P to argue you mentioned about three steps, synthase, hexokinase and transport, glucose transport, had they had all been implicated to be the roadblock, the step response for the insulin resistance, and so we were able to sort out which was rate controlling by measuring So if the block is at synthase,
Glucose 6 phosphate should build up and glucose should build up. If the block is at hexokinase, you should basically have lower G6P and a buildup of glucose. And if the block is a transport, there should be reductions in both glucose 6 phosphate and glucose. to a series of studies we found in not only these young lean insulin resistant offspring, but obese insulin resistant individuals as well as individuals with poorly controlled diabetes.
G6P, glucose 6 phosphate, and glucose are both reduced in the muscle cell, in vivo, in humans, implicating transport. That's where your biochemical block is. So the block is a transport. That's your target to fix if you want to fix muscle insane resistance. And the the corollary is these other steps are not good targets, drug targets to go after to fix insulin resistance in muscle.
This is the first abnormality we found in its transport and in these young, healthy 20-year-olds. And then the question is, what's wrong with the transport mechanism? And that led us into the world of lipid. Again, it's been known for decades that obesity associated with insulin resistance. That's why virtually every obese adult or child have insulin resistance. There are rare exceptions.
And then we basically found we developed a method to measure fat inside the muscle cell, and that was the best predictor for insulin resistance in the muscle and this block and translocation. Let's give people a quick primer on normal glucose disposal into a cell. So when the insulin molecule hits the insulin receptor on the surface. I believe it autophosphorylates itself, correct?
That then signals to insulin receptor substrate one, IRS one, inside the cell. So that sends a signal inside the cell, which also leads to a phosphorylation, which then signals PI3 kinase. It upregulates PI three kinase and And that basically leads to a GLUT4 transporter, which you can think of as like a big tube.
being shoved up to the surface of the cell across its membrane, and that basically passively allows glucose in. It is not an active transporter, correct? That's correct. Everything you said is spot on. Basically, up until now, we don't know where the breakdown is in that whole process. All we know is that something is impaired. in getting glucose in the cell, but in terms of
Is it there's not enough insulin that hits insulin receptor? Is there something wrong with IRS one, with PI3K? Is there something blocking the transporter? We're gonna have to figure that out still. But you've already taken two-thirds of this puzzle. off the plate by saying we know it's not downstream of that. That's correct. If you fix the transporter, that's where the roadblock is and that's the target.
The next set of questions becomes Why isn't insulin causing and as you point out, this translocation of the Gluc4 transporter to the membrane to allow glucose to come into the cell through facilitated transport down a gradient? So that's what we can talk about next if you want to. That's perfect. Can I share my screen with you at this point? You can and what we will do, Jerry, is we are gonna take everything that you are sharing with me and we're gonna turn these into
show notes that will be time stamped to this part of the discussion because While I guess people like you and I do tend to picture these things in our head easily, I think for many people it is going to be incredibly helpful to be able to actually look at some biochemical drawings. I benefit from this greatly. It's still not purely second nature to me. I like to think in pictures too. So as much as we can help the audience out with graphics, I think it'll be beneficial. So here's a cartoon.
I'll walk you through this and stop me if you have questions. This is a cartoon of a muscle cell. We went through how insulin normally works. Insulin binds to the receptor and everything, as you said, we're going to actually show this in this cartoon, binds the receptor. The receptor autophosphorylates becomes a kinase.
The key substrate for this kinase, insulin receptor kinase in muscles, insulin receptor substrate 1, which undergoes tyrosine phosphorylation, allows it to bind and activate the other protein, PI3 kinase, which Lou Cantley discovered, and that's a required step.
for translocation. So that's all been worked out. And somehow this is not working. This is broken in the insulin resistant individual. And again, these young 20-year-olds, the patient with diabetes, the obese insulin resistant individual.
And the question is, what's wrong? So I'm gonna share with you at least my view, which would explain insulin resistance in most situations of lipid induced insulin resistance, which I think accounts for I would say the majority of these patients I see who have type 2 diabetes
or who obese and insulin resistance or even these young mean insulin resistant offspring. And so this is the picture. So here, and it relates to fatty acid fat metabolism. Before I told you the other MR method that we developed Is actually something called proton NMR. And this is actually, most of your listeners are very familiar with, everyone knows about MRI, magnetic resonance imaging.
people go into a scanner and they get very pretty pictures of of an organ, brain, or some other organ for diagnostic reasons. And it's the same biophysical principles. You're you're basically getting this NMR signal from protons. And protons are the most abundant NMR visible nucleus in the body. And it's mostly water we're looking at. So when you're basically getting the same signal from protons, and mostly protons are water and fat. And so an imager
gives you this three dimensional reconstruction of proton density in water and fat. And that's what gives you the images. And again, we're doing biochemistry. So we're getting taking that same kind of information but actually looking at individual carbon atoms or phosphorus atoms or in this case protons lining the carbons and triglyceride. It's fat. So what I said using proton and MR to measure fat inside the cell.
This is different from fat outside the cell. So if you look at a steak and you see the marbling of fat in a steak, that's fat outside the muscle cell. What you don't see if you look at a steak is the fat inside the muscle cell. And using NMR we can actually discern fat outside the cell versus fat inside the cell. We can do this
in many organs and muscle started in muscle. And using this approach we found fat inside the muscle was the best predictor for this block and transport in all of our volunteers, young people, old people, children, sedentary individuals. Sedentary individuals, fat inside the cells, the best predictor for insulin resistance. And so this led us into the world of lipids.
we're keen to understand then if finding the lipid intermediate that might do this. And in studies where we took healthy individuals Perfectly normal sensitivity. We gave them an intralipid infusion, just raised plasma, fatty acids for three to four hours. and found that after three to four hours we can make them as insulin resistant as anyone with type two diabetes.
And others had shown that in addition to us. I mean this is we weren't the first to show this, but what we were the first to show is it's due to this block in glycogen synthesis. And it's the same block, it's that block in transport.
Just to be clear, when you deliver intralipid, that's intravenous lipid, as a triacylglycerol or diacyly? No, this is a triglyceride. This is a emulsion, a triglyceride emulsion. It's often given to patients for hyperalimentation when they can't eat you give us energy rich infusions. Just like TPN or something like that. It's used in TPN often. But what we also do is just a little low dose of heparin to activate lipoprotein lipase.
So all of a sudden then you can artificially raise fatty acids twofold, something from you know up to about you know one and a half millimolar and ask the question, what does this do? What does this have to do with does it ultra metabolism? And it has profound effects. So by increasing LPL expression Not expression. Activity. I did not know that Heparin activated LPL. So by activating LPL with heparin, cool trick to know. I'll keep that in mind.
you're gonna get more of that lipid into the muscle cell. You will raise fatty so what the heparin does is it causes activation of lipoprotein lipase. And that will then break down the triglycerides to raise fatty acids and more delivery of fatty acids to all cells in the body. Okay, so this becomes basically a quick vehicle by which you can deliver lipid directly into the muscle cell.
Exactly, where you can acutely change that. And again, you can't do this just by getting fatty acids. Fatty acid turnover is so fast you can't just infuse fatty acids to r significantly raise. So This is a way we're able to raise fatty acids specifically in vivo, in humans, and we do this in animals and it's that so it's a nice pharmacological way of asking the question, what impact does just simply raising fatty acids for a few hours have on metabolism?
And it's profound. It takes three to four hours before you see this, and then boom, it's you get very profound insulin resistance. And in our early studies, again, we showed using the same methods I told you about, measuring glucose 6 phosphate, measuring intracellular glucose, measuring glycogen synthesis. We found simply raising fatty acids for three to four hours blocks glycogen synthesis, profound insulin.
as I say, as anyone with obesity or type two diabetes. And it's due to the same an acquired block in transport, insulin activation of transport, both G six P and glucose are down. So that to us was a very important lesson because it basically changed the paradigm. Because prior to this, people workers, biochemists, you may know the name Philip Randall. He w did some pioneering studies in the sixties at University of Bristol.
and was really the first to say, hey, fatty acids may be toxic, maybe causing insulin resistance. and did studies in rat tissue, you know, cells, heart tissue, diaphragm muscles taken from rats in vitro, incubated it with fatty acids, and in vitro in the test tube induced insulin resistance. The mechanism that they postulated was that it was altering basically oxidation, the TCA cycle, citrate levels would build up and lead to inhibition of phosphofructokinase, which is a key glycolytic enzyme.
The prediction that Randall made was glucose 6 phosphate should increase. leading to inhibition of hexokinase. We were interested in that'cause we said, oh, fat in our hands is important. We're raising fatty acids and causing resistance. And we see this really strong relationship between fat in the muscle cell and insulin resistance in all of our subjects, obese. diabetic, young, insulin resistant individuals.
And so we wanted to see if his mechanism, Randall's postulary mechanism, translates to humans, because these were all in vitro studies done in tissues taken from animals. In a series of studies, we took again healthy healthy individuals raised fatty acids through this triglyceride and little dose of heparin infusion and found just the opposite to what Randall predicted. They got insulin resistance. which is what he would have said, but not through his mechanism. He said G six P should go up.
we saw it go down and we saw glucose go down. So it wasn't through inhibition of glycolysis, as he said, it somehow interfering with the insulin activation of transport. So and again, same rate controlling step we saw in all of our diabetics and obese individuals and prediabetic individuals. But just to be clear, Jerry, it caused hypoglycemia. The intralipid dropped glucose? No. Raising the fatty acids caused insulin resistance. Inability of insulin.
to stimulate glucose transport. Okay, okay. Yep. I may have misheard you, but okay. I'm gonna now fast forward. We then took these observations back to the bench, were able to replicate this in rodents, rats and mice and The power. Even though I'm most passionate about our human studies, um, I'm a clinical physiologist and I care most about understanding what's happening in
humans, the animal models allow you to really interrogate a biochemical process. There we can get tissue out. In humans I like to be non invasive with our MR methodology, but here sometimes you need to get tissues to measure activities, phosphorylation events, and and also you have the power of mouse genetics. You can knock genes in and out of mice.
to really rigorously test hypothesis. I should tell you one experiment before I move to this cartoon that we did in humans is we did biopsies in these humans when we raised fatty acids. and found this block in transport and asked the question, is a lipid intermediate, a fatty acid, a metabolite, interfering with insulin signaling cascade, which we just discussed, receptor and somewhere to PR3 kinase.
And what we found was indeed in healthy individuals just give glucose and insulin. You get activation of PHP kinase. This is the step you mentioned. This is the required step. For transocation. And in the follow-up studies, same individuals, we raise glucose and insulin and also raise fatty acids, and then we totally abrogate. Installin activation of pathrokinase. That study basically in humans, in the model we care about, is saying, yeah, somehow a fatty acid metabolite.
is leading to this block in insulin action somewhere between PI3 kinase and the receptor. So we've narrowed it down to that. I'll walk you through the steps that I think then are the biochemical metabolite that's mediating this, the lipid fatty acid mediator that's leading to this, and then the biochemical mechanism. Does that sound good? Yeah, that sounds fantastic. Here we have a cartoon of a muscle cell.
And my view, again, thinking about flux, it has to do with relative imbalance. So basically doing spokus lipidomics, we zeroed in on this metabolite, fatty acid metabolite called diaceyl glycerol. And yeah, I heard you mention that before. It's the precursor, it's the penultimate step in triglyceride synthesis, diacyl, two fatty acids on a glycerol backbone. This is a bioactive metabolite. It's been known for years to activate novel PKCs.
This is what we found tracked in our animal models with lipid-induced insulin resistance. Do high-fat feeding in a mouse or rat, get muscle insulin resistance, and it was this metabolite that tracked with insulin resistance. And then we did the lipid same type of lipid infusion we did in humans, simply raised. plasma fatty acids by giving triglyceride and heparin. We saw acyl coase go up, we saw DAGs go up. Right when DAGs w reached a peak, then we got
activation of novel PKCs, PKC theta and epsilon in the muscle. Then we link to this block an insulin action, which I'll show you in a second, at the level of the receptor and one step downstream of the receptor. The concept that I'd like to impart on you is it's this imbalance
between fluxes. So fatty acids are continuously being delivered to muscle cells. And we're gonna do the same thing if we have time to talk about the liver, because that's the other key insulin responsive organ. But we'll start with muscle. Fatty acids are being delivered either through fatty acids or even hydrolysis of triglycerides through LPL, endothelial barrier, delivering more fatty acids to the muscle cell.
When it's the flux of fatty acids into the muscle cell that exceeds the ability of the mitochondria to oxidize the fat. or store this fatty acids, acylcoasis triglyceride, you get net accumulation of diacoglycerol. This is a very important point. Triglycerides
are neutral. So I wanna emphasize this. So even though triglycerides often track with insulin resistance, we've dissociated it inside the muscle cell and liver cell from insulin resistance. It's a marker for DAGs typically tracks very well, but it's in a nurture. storage form of lipid. So triglycerides are not the culprit. We've dissociated in liver and muscle, but it's a pretty good marker if you can't measure the DAGs with mass spec.
Let's go back to that for a second. I want to make sure people understand what we're saying here. So a triacylglyceride or triglyceride, those two we use interchangeably, has this three carbon glycerol backbone with three free fatty acids on it. And that's the way that we very, very efficiently store energy in the most energy dense hydrocarbon in our body.
The DAG, by extension, has only two of those free fatty acids. What typically sits on that third carbon and what is it about that confirmation that renders the DAG, in this case at least, seemingly much more of a problem than the T G or T A G. Basically it's a hydroxyl group. a simple hydroxyl group. It's the two fatty acids of the DAG that sit into the bilayer, membrane bilayer, and then the hydrophilic hydroxyl group sits in the cytoplasm.
And that's what then will pull the novel PKCs to the plasma membrane. So that's the troublemaker. That's the troublemaker. Basically then when you get this imbalance between Fatty acid uptake versus oxidation in the mito versus storage as neutralipid, you get activation of these two novel PKCs in muscle, theta and epsilon. Theta blocks
insulin action at the level somewhere between the receptor and IRS-1 tyrosine phosphorylation. And epsilon, and we'll get into this for the liver, directly binds to the insulin receptor and then hits the receptor kinase. If we have t a chance, I'd love to share this with you and your listeners because this I think has important evolutionary mechanisms behind it. Why does this exist? And it's gonna be very important for survival during starvation.
But nevertheless, when both of these NPKCs in muscle are activated, you have reduced insulin tyrosine phosphorylation of IRS1, less PR3 kinase activation. And as we talked about, then less GUT4 translocation. So to me the real culprit, and we've been able to just quickly really test this rigorously, gene knockout, we've been able to inactivate isoforms and PKC theta, you get protection.
We've been able to block mitooxidation and you make these animals prone to fat buildup insulin resistance. We block fat entry into the myocyte, inactivate FAT4, they're protected. you overexpress lipoprotein lipase in the muscle, more fatty acid delivery muscle specific insulin resistance. And then finally if you rev up mitochondrial fat oxidation, let's say through uncoupling, overexpress UCP3.
in the muscle you get protection from insulin resistance. And all these track with DAGs going up or down with the insulin resistance or protection from insulin resistance. Let's talk a little bit about how we think. This is different in an active versus inactive person because the outset you said, look, when we're trying to find this in the youngest cohort of patients, these twenty-year-old, basically undergrads at Yale that we're gonna study.
We screen on many things, but an important thing we screen for is sedentary behavior. You mentioned that at the very outset, which leads me to believe that if you did a sampling across the cross country team, the crew team, you wouldn't find this phenomenon. So what is it about activity or the lack thereof that presumably points to this elevation of intracellular DAGs that kicks off this cascade.
Let me just show you. So this is where we talked about Revan and his hypothesis of insulin resistance and how What we wanted was to build on it. Because I'm going to answer your question about exercise but and I want to do two things now. I want to show you how exercise reverses this muscle insulin resistance. But I also wanna show you and your listeners why exercise in muscle actually will prevent fatty liver
and liver insulin resistance. I think that this is a useful segue. And so this is from Jerry Revens Mantine lecture in in nineteen eighty eight. And at that time people were still arguing whether insulin resistance was driving all these other things we see around the circle, atherosclerosis, hypertension, type two diabetes, polycystic ovarian disease, inflammation, or are these just common things clustering together?
So what we wanted to do was actually ask the question, what we see in these young twenty-year-olds, these volunteers, is the first thing we see is muscle insulin resistance. And maybe that's driving athrogenic dyslipidemia was going to lead to heart disease, high triglycerides, low HDL, and non-alcoholic fatty liver disease, by changing the fate of ingested carbohydrate from glycogen to fat.
So this is the distribution I was telling you about and g healthy young sedentary individuals, we're gonna get into exercise in a second. We simply take the bottom quartile one and four versus the top quartile. And we give them two high carbohydrate meals and we say, where's the energy going from that carbohydrate? How is it being stored? Getting at the very first question you asked me.
We can use our NMR to measure changes in fat storage in liver and muscle as well as glycogen in liver and muscle. And what we found then is you give them two high carbohydrate milkshake. And there's virtually no difference in the plasma glucose concentrations at at this late breakfast and lunchtime high carbohydrate shake. But you can see it is at the expense of severe hyperinsulinemia, is what we talked about.
The reason these young insulin resistant, as well as every insulin resistant person, is perfectly fine is the beta cells are pumping out two to three times the amount of insulin just to maintain euglycemia. So these beta cells are just being whipped. working really hard and that's why no one's diabetic. You're insulin resistant. That's why virtually every obese insulant person is normal glycemia'cause the beta cells are working so hard to maintain
And you can see that here. The other thing I want to point out is the insulin levels are uh give a number, you know, so normal maybe 100 at the peak and maybe 180 at the peak in the resistant individuals. But this is in plasma, the portal vein, what the liver sees is three times this. The liver seeing huge amounts of insulin in these insulin resistant individuals just to maintain normal glycemia.
We use carbon in MR to look at changes in muscle glycogen and liver glycogen. You can see, again, young, insulin-resistant 20-year-olds can't get glucose into muscle glycogen. Due to a block in transport, because they have increased ectopic fat in the myocyte, DAGs are up. No problem in liver. And then you look at the changes in fact. And this carbohydrate, this is change in liver triglyceride, it's up two and a half, two point three fold.
You put some heavy water, stable heavy water into the milkshake to track de novo lipogenesis. That's the conversion of glucose to fat, and that's also up greater than twofold. Quick question there. There was a very famous experiment. It's been so long since I've read it. I certainly know I spent many hours on it. It was by Mark Hellerstein, circa ninety-four-ish. And he looked at this question of how much carbohydrate could be converted to fat via de novolipogenesis.
And if I recall correctly, the answer was, at least from that paper, was not that much, but also I believe one of the criticisms. of that was that he was looking at an insulin-sensitive population. Am I remembering that correctly? Because what you're showing here would suggest the opposite, would suggest that an insulin resistant person is capable of significant de novo lipogenesis.
Everything you've said is correct. When you're thinking about de novolipogenesis, two things is you again, what conditions are you studying this? Is it after meal ingestion? Is it in the fasting state when a lot of people have measured this in the past? It's minimal and it makes sense. It only gears up. What substrate is taking in? And then depending on the type of substrate, you can alter this.
quite a bit. So it can be changed by simply putting more fructose, more glucose in the meal, by increasing the meal size. Mark's done beautiful work in the past. It is what it is. Those studies are what they are, but Clearly what we're learning here is just as you say, your DNL is significant. It's not the majority of the fat I think most investigators would agree the majority of fat synthesis in liver is occurring through a sterification, that is fatty acids
coming to the liver, getting incorporated triglyceride. But there is a significant importance for DNL. And again, especially if you track it chronically in patients who are continuously high carb feeding, especially high sucrose. High fructose corn syrup, we want to get into that, but fructose basically gets funneled into the liver, into the DNL pathway. It's ubiquitous. You can push DNL to be significant, and it is a significant contributor to metabolic fatty liver disease.
And it's upregulated with peripheral sensitivity. I think that this is the major message I want to give here is when you have muscle insulin resistance. Specifically, it will drive the liver fat synthesis by DNL. When you have that, when your liver is making more fat through DNL, it makes more BLDL exports. So plasma triglycerides go up and HDL goes down.
So what I find interesting about this before you go further, Jerry, is this is all from the two thousand seven P NAS paper by your wife, actually, right? Kit Peterson. Yeah, Kit Peterson. So what I find interesting about these data is that these patients were euglycemic. I mean, that to me is the staggering piece of this. These patients are still potentially a decade away from seeing an interference in glucose homeostasis.
They're a decade away from their doctor saying, hey, your glucose is a little higher than it should be postperandulae, never mind even at the fast. And yet they're already seeing an 80% increase in triglyceride, which I just want to sort of talk a little bit about this clinically. Most laboratory assays will say a triglyceride level of one hundred fifty milligrams per deciliter is considered normal.
Well, we don't say that. In our practice, we view anything over a hundred as abnormal. That's a red flag. And if your trigs are more than two X your HDL cholesterol, that's a very big red flag.
although most people would accept triglycerides of three or four, if not five times above HDL cholesterol before the sirens would go off. And yet when you look at these patients, again, euglycemic you see a difference of approximately, you know, 100 to 105 of the trigs in the insulin resistant to 60 in the insulin sensitive. So it's all kind of right here in front of you, sort of in a way that unfortunately just doesn't get appreciated.
But it's the more intense stuff that's mind-boggling to me, which is the two and threefold difference we see in de novolipogenesis hepatic. Synthesis of fat, impaired hepatic glucosensitivity. And I guess it speaks to the point you made earlier, Jerry, which is. When the portal vein amplification of insulin differences is as big as it is, it becomes basically a magnifier of everything we're seeing in the periphery.
Exactly. Yeah. And our normative data, in my view, we need to reset. What we consider normal is to me this is when we look at our insulin sensitive, that's what our normal should be and guiding us. You asked about exercise. That's something we're quite passionate about and I wanna kind of see tell you how that fits in here. So again, conceptually
Here we have a normal person ingesting carbohydrate. First question, how is this distributed? It's in glycogen. This is where you want to store your ingested carbohydrate. It gets stored in glycogen and liver and muscle. And again, this is one quarter of our young, lean, healthy volunteers are insulin resistant. And again, if you're overweight or obese,
you're there already. Because these are lean individuals, and that's still one quarter of the population. You can't get that ingested glucose into glycogen due to this block in transport, due to the block in DAG PKC inhibition of insulin signaling. It's diverted to liver. You have that insulin in the portal vein, that's three times periphery, it's up to five, six hundred microunits per mil.
That turns on SRBP1C, the master transcriptional regulator of triglyceride synthesis, gears up all the DNL enzymes. So you have increased DNL. That leads to this increase, we just reviewed plasma triglycerides, this reduction in HDL. This is gonna set these healthy individuals up to athrogenic dyslipidemia, heart disease in their forties and fifties. With time it's metabolic associated fatty liver disease. Now, and again, most common
Cause of liver disease now in the world. It's now leading cause of NASH, leading cause of liver fibrosis, cirrhosis. And stage liver disease and gonna be liver cancer. So it's all gonna be metabolic driven and from that hyperinsulinemia, in my view. So exercise. Can we do anything about this? This hypothesis is right, we can test it. And so you asked about exercise. So this is a study we did some years ago, published in the New England Journal.
Took these young insulin-resistant offspring, and this is with parents with type 2 diabetes, and the Jocelyn group. did a really nice study. They found that if you have two parents with type two diabetes and if you're insulin resistant, that single parameter is the best predictor for whether or not you would go on to develop type two diabetes. So we've tried to study these individuals with our methodology.
extensively. And John Luca Persagan, who did this study when he was a fellow with me, Took these and eventually just studied them in the basal state, shown here that, you know, again, in the he's young, insulin resistant, again, everyone's here is lean, non-smoking, no medications, they're in their twenties and thirties, BMI 23, 24 to factor out.
Obesity, confounding of factors of obesity, medications, smoking, other things. So young, lean, healthy individuals, but just parents with diabetes, insulin resistance. You study them and in the basal state take up less than half the amount of glucose in muscle and it's due to a block in transport. So same thing as I've gone on and on before in the diabetics and the obese individuals, this block in transport.
And we asked the question: Does exercise can we bypass this abnormality? And the answer is yes. So here you can see this was after six weeks of Uh being on a stairmaster three fifteen minute bouts at about sixty five percent MVO two max. And here we're normalizing insulin-stimulated muscle glycogen synthesis. And we've usually measuring glucose 6 phosphate, we've opened up that door of getting glucose into the myocyte.
And I think molecular explanation for this is this protein called ANPK, which we can talk about. gets activated with exercise. And that has been shown to cause vor translocation independent, independent of peatric kinase. And so we're kind of short circuiting that block with exercise. To test our overall hypothesis, does muscle insulin resistance drive fatty liver?
and DNL and high triglycerides, we took these young insulin resistant individuals and we showed, John Lucas showed in that New England Journal study, even a single bout Forty five minute bout was sufficient to open up the door to glucose, cause that glut four translocation. And Rasmus Raball, when he was a clinical fellow with me, did one single bout in these same individuals I showed you before, insulin resistance in muscle, the ones had high triglycerides, low HDL, and prone to increased DNL.
With the single bout, we're able to show that that same ingested glucose would lead to more glucose deposition as muscle glycogen. And we got significant reductions in de novo lipogenesis, significant reductions in liver triglyceride. I just want to make sure I understand that and it's relevant to another question I have about the difference between insulin dependent and independent glucose uptake. So Do we know if that single bout of exercise Which particular piece of the pathway got
released. Did it have some direct effect on the root cause the DAG or some of the kinases downstream? Was it even further downstream at the very last step where the transporter gets released? Like where was the actual bottleneck alleviated with that single bout of exercise? I can speculate. In these were human studies, I can tell you that we open up the door, we measure glucose 6 phosphate in them and and that's
goes up. So we open the door for that defect in insulin stimulating transport is now reversed. So glucose transporters are in the membrane, glucose is coming in. What I can't tell you is whether or not we've altered DAGs and we get in improved insulin signaling at the level of the receptor and IRS one.
and or is it just AMPK causing this Glutefor translocation? If I had to speculate, I would think most of it is through the latter. We were simply with an acute bout causing AMP K induced GLUT4 translocation, which we know happens independent of PIPinate. That's established. So we're short circuiting. We're just causing GUT four right at all the lower mechanisms to get to the membrane. So we fixed the block in insulin action. I think though
With chronic exercise therapy, we're going to be doing both where we get melt away the lipid and DAGs go down. So we have improved insulin signal as well as more AMPK induced Glu4 translocation. Yeah, I'll tell you just I think I've even discussed this on a previous podcast. I've had a couple of patients with type one diabetes that I've taken care of, not many, but in the phenotype of patients with type one diabetes where there is a significant amount of exercise.
specifically sort of modest intensity aerobic exercise. So a person who is, for example, doing brisk walking, very brisk walking, sort of to the tune of four miles an hour, an hour to two hours a day. These patients with type 1 diabetes can be virtually free of insulin.
and maintain reasonable glycemic control. So they can walk around with a hemoglobin A1C of six percent, using maybe 12 units of insulin a day, and obviously restricting carbohydrates. But again, it suggests I say this having watched them change the intensity duration of the exercise, that it seems that that exercise becomes a spigot to how much. Glucose they can dispose in their muscle seemingly without insulin. It's almost like a total bypass of the system to
Which again, I think, to your point is chronic. I don't think this is something we see acutely. I obviously can't comment on it. The first time I saw it, which was probably about six years ago, it really sent a light bulb off, which is Imagine now being able to maximize both insulin dependent and insulin independent glucose uptake into a muscle, that really becomes a powerful tool to combat all of this sort of metabolic dysregulation.
That's what AMPK does is insulin dependent glucose uptake. And I can see in combination with reduced carbohydrate consumption, less coming into the circulation and whatever little comes in is taken care of through AMPK, insulin-independent GLUT4 translocation. So that fits. Before we go to the liver, and I do want to actually talk about how all of this works in the liver.
I want to go back to one other thing that you very briefly touched on, which is the evolutionary Explanation for some of this. That would be best done, if I might say, with the liver. Okay, great. Let's do it. Because I wanna understand this, yeah. That's kind of fun. So let's now turn so I I've kind of walked you through at least my thinking about insulin resistance, why it's so important for not only diabetes, but so many diseases. I've shown you the physiological
cause for insulin resistance in muscle, can't get glucose in the glycogen. I've shown you that block is a transport, and then I've given you a molecular understanding of how that insulin resistance in muscle happens. My view is lipid disoglycerol is blocked leading to activation of a novel protein kinase C epsilon theta blocking insulin signaling. Okay. So let's now and then I've shown you how muscle insulin resistance can lead to fat accumulation in liver, atherogenic dyslidema and fatty liver.
Now we know fatty liver is what then leads to insulin resistance in the liver. And so I want to take you through the molecular basis for how fat and liver causes insulin resistance. And it's pretty much What's nice now that you understand muscle lipid induced muscle insistance, it's pretty close to the same story
in liver. So here's a cartoon of the liver cell. But is the direction of causation, Jerry, in the order in which you're telling the story? In other words, is the hyperinsulinemia as a result of muscle insulin resistance Let me clarify that. Muscle insulin resistance, which leads to peripheral hyperinsulinemia, which is accompanied by portal vein hyperinsulinemia, which leads to what you're about to tell us. Is that the order in which you think this occurs?
I do. As I say, this is what we see in our volunteers as we march through the progression and different stages. We don't see liver abnormalities in these young twenty year olds. It's all muscle and maybe a little bit of the fat cell, which we'll come to at the end, but It's the muscle, there's no alterations in the liver until they get fatty liver. Once they get fatty liver, then we see both. insulin resistance in liver and insulin resistance in muscle.
A very important distinction between humans and rodents. We've studied both models quite extensively. Rodents develop insulin resistance in it the reverse direction. They get liver fat first, liver insulin resistance, and then muscle.
Most of the studies on this are done in rodents. It's a very important distinction in terms of the progression and very different humans versus rodents. And we can talk about similarities and differences if you want, but we're going to focus mostly on humans for this talk. And that makes total sense. So it is again, it's peripheral IR, hepatic IR, hepatic consequences, which then basically amplify it.
That's my belief, yeah. And again leading to this beta cell compensation, compensation, and then again something when you get both muscle and liver insulin resistance and x increased glucose production by liver. then something happens to the beta cell and that's when things really start to spiral where you have very profound hyperglycemia, fasting of post grannial. Here's the cartoon of the liver cell.
And again, glucose transport's not ray controlling, as you know, in the liver cell. Glucose just diffuses in through GLUT2 transporters. And the insulin again binds the receptor, same thing, autophosphorylation. The key intermediate there in liver is IRS2, undergoes tyrosine phosphorylation, piath kinase, just as you did in muscle AKT2. And in liver what happens is
You have a few things. One not shown here is glucokinase, translocation, and that we've recently shown is probably very important for rate control, getting glucose into the hepatocyte. You also get activation of glycogen synthase. And more glycogen synthesis. And then you have this phosphorylation of Foxo which is a transcriptional regulator and that then is excluded
from the nucleus and then down regulates then glucanogenesis through a transcriptional mechanism. And if we have a chance, I'd like to come back to this because we have some interesting data that speaks to really how insulin's inhibiting this key process. So let's now just focus on how lipid causes insulin resistance in liver. Same metabolite, it's the disoglycerols.
They go to activate epsilon. That's really the major isoform of PKC, novel PKCs in liver. And work by Varman Samuel when he was doing his PhD with me in a series of studies. Varman showed that epsilon binds to the insulin receptor and directly inhibits the receptor kinase itself. And that then leads to downstream abnormalities.
What I want to share with you now, which I think, and again, it gets into this evolutionary basis for insulin resistance, which I think your listeners might find interesting, is how is epsilon inhibiting the receptor kinase? We worked on this, Jesse Reinhard and Max Peterson. He was an MD PhD student with me. We did untargeted phosphoproteomics, and what I'm showing here is the catalytic
domain of the insulin receptor. Yeah, I can just describe it for the listeners. It's a loop. You can picture it as a door. over the pocket for the catalytic domain of the insulin receptor and this door is closed. IRS one, IRS two can't go into the pocket for tyrosine phosphorylation. When insulin buys the receptors. These three tyrosines, the eleven fifty eight, the eleven sixty two, and the eleven sixty three become phosphorylated.
That opens the door, that loop flips out, and then IRS1, IRS two go into the pocket and undergo tyrosine phosphorylation to get the rest of the cascade going. Using untargeted phosphoproteomics, we were able to show Jesse Reinhardt, who is our collaborator and Mass Spec. Maven identified using purified receptor, purified PKC epsilon, that when you add activated epsilon to the receptor, you phosphorylate this threonate.
And that got us very excited because, golly, that's one amino acid away from these two tyrosines that are required for activation receptor. may be doing something important. And so the other thing that got us excited about, and here's getting into evolution, is the sequence of the catalytic domain for the receptor. And it's been conserved All the way from humans down to fruit flies.
Those three tyrosines, same position, and that threonine that sits right between the two tyrosines, 1158 and 1162, has been conserved all the way again from Homo sapiens down to Drosophila. through evolution, if something's important, it usually hangs around. That's a long time.
So to prove this, we very simply we did some genetics. Again, that's what you can do is you can knock A glutamic acid replace that threenine with glutamic acid, mimic a phosphorylation event, and that kills the kinase activity. You can mutate the threenine to an alanine so it can't get phosphorylated, and then you have protection in vitro from epsilon-induced reduction in IRK activity. And then you can make the mouse. And so here in this paper, we made mice where we replaced
The threonine in that key position, the eleven this is the mouse homologue, the eleven fifty is the homologue for the eleven sixty in humans. So all the threonines are instead alanines. And I won't get into the data other than say the mice are perfectly normal, normal chow, normal instant sensitivity, nothing that you know normal size, normal growth. But when Mac
fed these mice a high fat diet, the wild type mice get profound hepatic insulin resistance. And this we see and everyone else on the planet sees you feed mice high fat diet. Even for three to days they get profound FAT accumulation, DAG accumulation, hepatic insulin resistance. Does it have to have sucrose in it as well, or just fat?
Doesn't need to be. You can make it worse if you add a little sucrose. They like that in the drinking water and they have exact even more fatty liver if you put sucrose in the drinking water. But This is just with fat alone, but it's even more greater when you put sucrose or fructose or whatever sugar you want in the drinking water.
And here then you can see when you simply mutate that 3-neutrin aline, now you have perfectly normal hepatic insulin sensitivity as reflected by insulin's ability to suppress hepatic glucose production. And this is despite the same amount of liver fat, same amount of liver DAGs in the liver.
This tells us that that single amino acid is doing something very important in terms of mediating lipid-induced insulin resistance. And this actually just came out this last week, this paper now, just to summarize where we've now shown that there's different isoforms. We didn't get into this, into the isoglycerol. And it really matters which isoform it is and w what compartment it is.
Just to summarize this paper that just came out in cell metabolism, we were able to show by measuring the three different stereoisomers of disoglycerols, it's really the SN12. and measuring these different isoforms in five different intracellular compartments, the plasma membrane, the cytosol, lipid droplet, ER, and the mitochondria, it's really specifically the SN1 to isoform in the plasma membrane that's important.
If you just measure total DAGs, you may easily miss this. We learned that this recent study and that we we showed both that PKC epsilon is both necessary and sufficient for this process by doing the knock-in and over expression. I just wanted to basically touch on the question you asked me about. Why do we have insulin resistance? Why should it exist? And the reason I think it exists is it's protective for us during starvation.
When you starve, this is true pretty much in all mammals, mice, rats, and humans. When we starve. We get fatty liver. Here in this study, this is Rachel Perry's paper in cell from a couple years ago. Take rats, just starve them for 48 hours. You have increased liposis, more fatty acids delivered to the liver, hepatic fat accumulation, DAGs we show go up, SN12, PKC epsilon translocation.
and insulin resistance in liver. And the main thing that insulin does in the liver is it promotes glucose uptake and storage as glycogen. When you think about it, That's what you want turned off during starvation because during starvation glucose is a very precious molecule and you want to preserve this in circulation for the CNN. which is critically in need. It's really the major source of energy
for the CNS. And so by promoting hepatic insulin resistance, we're promoting glucose in circulation for basically the CNS to operate. And so That to me is why that three inning is preserved all the way from humans to fruit flies and I just wanted to show you this cover of nature, this Mexican cave fish. It's a fun story because after our paper came out, this little fish made the cover of nature. And what was so fascinating about it is so these little fish, they live inside caves.
They are spend most of their life starving. The only time they are able to eat is when something smaller than them swims in front of the cave and then they can reach out and grab it and pull it back into the cave and gobble it up. And these workers who studied this Mexican cavefish found this cavefish had a mutation in the insulin receptor, had profound hepatic insulin resistance. And they also went on to say this was important to allow them to survive.
In my view, insulin resistance was a protective mechanism throughout evolution. that allowed us to survive all species during starvation, which was probably the predominant environmental exposure we've had for the last many, many millennia.
And it's only in recent years, recent decades that now we're in this toxic environment of n overnutrition and it's when these same pathways now are going the opposite direction, promoting Disease by doing what they were at one time was protective, and now they're actually being told metabolic disease that we just discussed.
So I wanna make sure I can unpack this a little bit. So I wanna start in the muscle'cause I think it's easier And again, we'll even talk about it in humans, which means we can do it on a sort of different timescale, because obviously forty-eight hours of fasting in a mouse is a seismic fast, a near fatal fast.
But let's say forty eight to seventy two hours of fasting in a human We still would expect to see significant muscle insulin resistance, and there would be a great reason for that evolutionarily, because
you would want to make sure that as much glucose as possible in that circulation, which by this point is all coming through hepatic glucose output, is not being quote unquote wasted in muscle glycogen synthesis. To your point every gram of gluconeogenic substrate that's going through the liver and then coming out the liver should be preserved for the brain, because even Cahill's studies showed that after forty days of starvation,
humans were still getting about forty percent of CNS energy from glucose, the remainder from ketones. So glucose never went away as a substrate for the brain. So I think I have a handle on the muscle side of things. I'm still struggling a little bit to understand the physiologic consequence of hepatic insulin resistance and how that feeds into what I think should be an environment that says, figure out a way to make as much glucose
for the CNS as possible? Why does more fat accumulation in the liver make it better served to protect the brain? So first of all let me step back. So both organs during starvation, both liver, even though I focused here on liver, muscle will become insulin resistant also through increased circulating fatty acids through the mechanisms.
We talked about DAGs building up PKC theta. So insulin resistance in all organs are going to preserve glucose for the CNS. I was just focusing on uh the three here in liver because that's where epsilon was taking us. To understand the liver, I want to just take you to another cartoon because you're asking a very important question about processes, about regulation, how insulin works.
in liver. And I think to do this, let me just step back. The conceptual view, again, this is a cartoon I always like to show. How does insulin work? This was from twenty years ago when I was first studying it, maybe thirty years ago. Insulin binds to receptor, magic happens, something happens, then you have an effect. And so Even though insulin's been since its discovery, we're still trying to really understand what's happening in different tissues, how it works.
And getting surprises. So this is the canonical view we just went through of how insulin works on liver, it binds the receptor, it activates the cascade to promote glycogen synthesis and turn off glucanogenesis. And what we're finding is This simple view doesn't explain many things and I think needs modification. Especially in terms of insulin regulating glucanogenesis, this process that is required
to keep us alive during starvation. Without gluconogenesis, we're not gonna wake up in the morning because it's glucanogenesis that supplies glucose for the CNS while we're sleeping. And certainly during starvation, without this process, we're in trouble. I don't think that can be overstated, by the way. Let's go back to what you just said. We couldn't survive by my calculation, Jerry, we'd have a hard time surviving ten minutes without gluconeogenesis as a species.
Well, I'll modify that a little bit. I'd love to hear you state the importance of glucanogenesis. No, we know clinically you can. And again, from the lessons learned from gene knockout, you know, unfortunately, the patients with inherited disease, Von Gerkey's disease. As you know, patients who don't have glucose six phosphatates, the last key step getting glucose six phosphate out.
We do know that can be compatible with life. We have patients with glucose 6 mossetase, and the way we keep them alive is just continuously to feed them. Yeah, that's my point. Without continuous glucose feeding, your lifespan would be measured in minutes to hours without gluconeogenesis to regulate glucose homeostasis.
It's critical for life function. We're on the same page. So let's just talk about then how it's thought to operate and regulate it. It's also important to be able to modulate it. So we eat a meal and we have to suppress glucanogenesis. Otherwise glucose would go up to four or five hundred after eating a carbohydrate meal. So it has to be a process that's turned on, turned off. And not turned on too much, you know, in terms of diabetes, because that's what dries fast in hyperglycemia.
Traditionally, pretty much the major textbooks, physiology, biochemistry, Insulin is thought to be turn off glucanogenesis through transcriptional mechanisms. And again, this is the SFOXO phosphorylation by AKT, exclusion from the nucleus. Then you get downregulation of Pepsi cake, excuse me, and six phosphatase. FOXO is the transcription regulator for these downregulations.
The problem with this view, and again, there's some beautiful molecular biology, and I'm not wanna deny that this doesn't happen, but the problem with this being the predominant regulating mechanism is threefold. One is you can knock out AKT or FOXO and give insulin to the mouse, and you can still turn off glucanegenesis in a fasted mouse, which is totally dependent on glucaningenesis. That speaks to the fact you don't need these key insulin signaling pathways to regulate glucogenesis.
The second thing in terms of its role in mediating fasting, hyperglycemia, and diabetes, is We got liver from patients with poorly controlled diabetes. So when patients go in for a ruin Y or bariatric surgery, the surgeon can take a little piece of liver under direct visualization, so it's very safe. and give us enough liver to we can do actually protein measurements and enzyme measurements of Pepsi K6 posters. Not just message, but actually the proteins themselves.
And to my surprise I thought all these enzymes from everything I was thinking about biochemistry and at least what I learned and when I was lecturing medical students, I expected Pepsi K and six phosphatase and Fructose one six by phosphatase all to be upregulated two to three fold. in the poorly controlled diabetic that was undergoing ruined by pipast surgery compared to the non diabetic.
And we found no relationship between protein expression of these enzymes, glucogenic enzymes, and at least fasting glucose and insulin and history of diabetes. Finally, when you develop methods, the flux methods we won't get into to actually quantify this flux of glucanogenesis, which has not been easy to measure, by the way, but we have methods now, they're very good to measure this flux. We can turn off lupinogenesis within five minutes.
And that's much faster than you'd expect from transcriptional translational mechanisms. Just to kind of talk about how glucanogenesis, this is the glucanogenic pathway lactate to glucose. You can have transcriptional regulation, you can have substrate regulation. So glycerol we've shown from lipolysis, there is no rate control. The more glycerol that comes
From fat breakdown in the fat cell, that fluxes to the liver comes right out as glucose. There's no rate control, it's just all substrate-driven. Redox we've shown in the liver cell regulates glucanogenesis and this in a series of studies that Anilla has done. That's how I think metformin works, and we can talk about that if you're interested.
But finally, I want to emphasize is this allosteric regulation of glucanogenesis by acetyl-CoA. This had been known for decades to be a regulator of pyrovicarboxalase. and had kind of been forgotten because it was very hard to measure and no one looked at it in vivo because it's hard to measure in vivo or especially in a diabetic situation.
We said, well, wait a minute, let's go back and look at acetyl-CoA. We developed the methods, tandem as spec methods, very sensitive, very specific, to do this in freeze clamp tissues from animals with varying degrees of Diabetes hyperglycemia. The bottom line is found a very robust relationship between acetyl-CoA, which is as you know, the end product of beta oxidation. Take fatty acids and break them down through beta oxidation, the end product as before it enters the TCA cycle.
And there's this very robust relationship, just all these different studies, but basically every study we do, we give insulin, we get suppression of acetylchoid. This explains how insulin acutely suppresses glucanogenesis when diabetic models, when you have increased glucanogenesis, it's twofold increases in acetyl-CoA, but it perfectly follows rates of glucanogenesis, which we quantify
track perfectly with concentrations of paticacetoquate content. I just want to take you how insulin normally works in the liver cell. and then how it becomes dysregulated in diabetes. And this is going to answer your question about How do we distinguish insulin promoting storage as glycogen yet keeping glucanogenesis going for the brain? So this is very important to answer that question. So in my view, Insulin binds the receptor and it has direct effects.
through the receptor. That is mostly to promote glucose uptake and storage as glycogen. The effects on gluconeogenesis, the process that keeps us going during starvation, is really mostly regulated not through the receptor in liver, but it's through its effect on the fat cell. in the periphery. In studies we've done in awake rats, and we're translating this to humans, it's really insulin putting the brake on peripheral lipolysis.
Less fatty acid delivery to liver, less generation of acetyl-CoA. And we've shown this the more fatty acids that flux the liver. track almost perfectly with acetyl CoA content, less pyrovic carboxylase activity, and again there's about 10-15% of this glucanogenesis is simply coming from less glycerol from lipolysis to liver through substrate push. So you have two very different processes here. One is glycogen synthesis. That's what the receptor is doing in the liver.
Glucaneogenesis is mostly ninety percent I would say. There may be a little bit of intrapatic lipolysis regulation, but mostly through its effect to put the break on peripheral lipolysis. And this model, by the way, will explain In my view, uh the explanation for all the controversies of insulin action that have been described through the last decades in mice, where you knock out AKT in the mouse, insulin still work.
you do things to the periphery, fat cell, and you affect glucose metabolism, glucanogenesis. All these Studies that appear to be conflicting can be explained if you use this model as a template to understand insulin action. And again, I have short-term fast and long-term fast. This is important, species differentiation.
And as you pointed this out, Peter, even after an overnight fast, boom, all their glycogen's gone. Very different from humans. Humans hold on to their glycogen like dogs, probably for ten minutes. Two days. We've done these measurements with starvation in humans. We've shown that it takes about two days to deplete liver glycogen. When you have glycogen in liver, it's really these direct effects of insulin on liver will predominate.
But as you move to the fasting state, so in a mouse after a 12 hour fast or longer, and in a human probably have to go twenty-four or longer fast, then it's really insulin, these indirect effects will predominate. And this will also explain all the controversies in Dogs, Sherrington, Bergman in terms of direct, indirect. They've each published a dozen papers on going back and forth which predominates. This mechanism would explain, I believe, all of those findings.
And then I just want to now show you how I view the dysregulation in diabetes. So now typically on the background of obesity, which is what happens in most of our diabetics, although you have lean individuals who also have this. You have expanded fat stores in the periphery, but now you have insulin resistance in the fat, so insulin can't put the break on lipolysis and we can talk about that mechanism which we're now working on, but it's going to be very similar in terms of liver and muscle.
But you also have this component of inflammation. This has been described by many, many individuals. You get crown-like structures, macrophages, move in, they release TNF alpha, IL6.
And what we were able to discern a lot of people would argue it was inflammation. If you go back to the insulin resistance literature ten, twenty years ago, everyone was discussing inflammation Circulating cytokines, TINF alpha L6, resistant RBP, circulating factors that were released from inflammation driving insulin resistance.
What we found is, again, you can dissociate inflammation from insulin resistance. That's what I spent the first three decades of my life doing, showing that just ectopic lipid. DAGs would drive insulin resistance independent of inflammation, but the transition from just insulin resistance in liver and muscle. too fasting hyperglycemia depends on inflammation. And it's through this mechanism where now you have localized inflammation in the fat cell.
TNF alpha, IL6, I'm sure there's other things, will promote increased lipolysis in the fat cell. More lipolysis, more fatty acid, delivery to liver. DAGs go up. Epsilon gets activated, you block insulin action, so you less glucose being taken up into glycogen. This is what happens in virtually most patients with fatty liver disease. But again, what takes you to fasting hyperglycemia? is that
And that's where acetylcoA goes up. And again, now your rates of lipolysis when you measure turnover, not just fatty acid concentrations, but turnover, pelmitate turnover production and glycerol turnover. It's up twofold. This increases acetyl-CoA concentrations twofold. This activates pyruvicarboxalase activity and flux twofold. And then in addition, your glycerol delivery to liver is up twofold, and now your rates of glucanogenesis are increased.
Twofold. And this is now what's driving fasting hyperglycemia in every poorly controlled type 2 diabetes. It's this glucaneurgenic process. that we've shown using many, many methods and others have shown this too, this is what now is driving hyperglycemia in type two diabetics. Okay. I have several questions, Jerry. First. These adipocytes that are undergoing lipolysis, these are peripheral adipocytes. Is that correct?
Yes, you can have situations where even fat in the liver is probably contributing to this, especially in the lipodystrophic individual that has no peripheral fat cells. So that under conditions the liver fat is playing a role, but most of it In most of, you know, I would say garden variety when I see it's going to be peripheral lipols. So when we think about an insulin resistant, obese person with metabolic syndrome, so this is what, twenty percent of the US population, maybe even more.
We've clearly established they are insulin resistant in the muscle. We've established that they are insulin resistant in the hepatocyte. They are obese. So would we still say they are insulin resistant at the fat cell, or would we say they are insulin sensitive at the fat cell because they are correctly
undergoing lipogenesis in the fat cell. They're at least taking up a sterified fat, and they're presumably impairing lipolysis, which is why they retain adipose cell mass. In other words, there's a the flux through the fat cell is negative. They're holding on to fat, correct?
Yeah, but I think and this is a question a very important question we're going to next. I would still predict if you do careful studies of measuring rates of lipolysis by definition they will have insulin resistance in the fat cell. And that's because the reason they're doing everything you just said, they're holding on to fat.
they're not happy about it, the doctor's not happy about it, is because it's at hyperinsulinemia. So their insulin concentrations are two to three fold. So again, their curve is right shifted. Insulin's doing the thing, but if you brought them down to normal levels of insulin, then you might see more lipolysis and other things. So I think if you were to do those studies, and they've been done, there is peripheral insulin resistance.
But then you superimpose in addition and I'll just say I'll share with your listeners, we're finding actually the same mechanism that we have in liver and muscle, and we're seeing this in many other tissues too. In the fat cell, the disoglycerol epsilon pathway is also accounting for this defect in insulin action in the fat cells. So it's gonna actually be a common mediator and again y most of the fat, of course, in the fat cells in the lipid droplet. So again, the plasma membrane
Disoglycerols that lead to epsilon activation in the membrane and the fat cells. And we're seeing the same thing. And we see those same mice that I showed you before, the IRK NOCA mice are protected from lipid-induced fat insulin resistance. On the fat topic, we've talked a lot about the intramiocellular lipid. You've distinguished it from, say, marbling or fat between cells. One thing we haven't spoken about
that clinically gets a lot of attention is visceral fat. So you alluded to doing an MRI. So we do a T one weighted image of a person on an MRI gives us a beautiful resolution anatomically of what's happening. And you can see the difference between a healthy person and an unhealthy person and one of the most glaring differences between people on that type of proton imaging is the amount of fat.
that is inside the fascia. So you have subcutaneous fat that may not be aesthetically pleasing, but more importantly, when you go inside the corset of fascia, You have some people that will have a heavy ring of fat around their kidneys, their spleen, their liver. We call this visceral fat and the association between this amount of visceral fat
and poor health is very well understood, whereas there seems to be very little association between subcutaneous fat and poor health. How does that visceral fat identification square with the intralipid myocellular component that you've described so elegantly at a cellular level. In my view, and everything you said is correct, it's up to you if you're gonna store fat somewhere, that's the best place to store it. You certainly don't wanna keep it inside liver and muscle cells.
In my view, and again studies have been done to look at the visceral fats, and it's very clear it is again a very apple shaped people have visceral fats, a very good predictor of insulin resistance. It's really more of a marker for intrapatic fat. So any time if when you're doing your imaging, if you just look at the liver two, they're gonna correlate one to one ninety-nine out of a hundred times. So what you're really doing there
is a marker. Now it's the visceral fat will also pour fatty acids into the portal vein, presumably, and again fatty acids delivery portal veins probably going to lead to increased acetyl-CoA. You know, again, will contribute some degree. To me, the major abnormality is really the fat inside the apatocyte, more importantly, the acetyl-CoA within the hepatocyte. I want to give one example that makes this point clearly, at least to me, the lesson I learned, and that's lipodystrophy.
And as you know, that's a situation where there is no fat, no sub Q fat or visceral fat. These patients. have no visceral fat, huge livers, hepatomegaly, chalk full of fat and liver, and again diabetes through these mechanisms, acetyl-CoA drive and glucone genesis. And that's independent of visceral fat. So that shows you you don't need the visceral fat at all to drive this. It's fat in the hepatocyte. If I had to pick two molecules that are driving metabolic disease, it's Acidal Coei.
driving perverticboxylase and again the disoglycerols activating epsilon. And again, it's the epsilon that drives insulin resistance, no diabetes, no hyperglycemia. Then it's this accelerated glucanogenesis. through this mechanism that's taking you from just pure insulin resistance to fast and hyperglycemia and diabetes. So let's again pause there for a moment and unpack something very profound.
If we've just established that the accumulation of liver fat is effectively the hallmark of death to come, and you just said acetyl-CoA and DAGs are two of the biggest culprits, Well acetyl CoA, of course, is abundance of nutrient on some level, which speaks to something you said earlier. You take a patient with type two diabetes, put them on twelve hundred calories.
a day, by definition, that has to lower acetyl-CoA. That immediately is going to improve things, which it does. Whether that's sustainable indefinitely, we can discuss. And of course we've already established where these DAGs are coming from. Again, I wanna pause for a moment on that because I think a listener of this right now is gonna say, Guys, you've lost me, okay?
They don't know the difference between Pepsi K, GSK3, AKT2, PI3 kinase. I don't think you have to know those things. I think what you have to understand is that abundance of nutrient is a relative term. It's not an absolute term. An athlete versus a secondary person has a very different amount of what that abundance looks like. I think we've also discussed that not all nutrients are created equal. You've alluded to it already that sucrose and fructose disproportionately prime the liver for this.
And then of course we're dealing with carbohydrate metabolism. This is perhaps an interesting time to also start talking about. both the modifications that we can make, because again, when we start to think about you've talked about Western diet and sedentary behavior a lot. So there's no doubt that there is and are environmental triggers contributing to these epidemics. which largely began here in the United States, but we have Fabulously spread to the rest of the world. And then of course.
There's a whole pharmacologic side of this. I would like to revisit the Metformin question. I think it's a very interesting question. Metformin works presumably by sort of weakly poisoning the mitochondria at complex one, that would lead to a redox change of NAD and NADH, which goes back to something you talked about.
But as of this time at least we don't really have many exciting compounds in the pipeline for NAFLD, which as you also alluded to, in about ten years is going to through NASH and cirrhosis be the leading indication for liver transplant in the United States, something that when I was in medical school accounted for less than two percent of liver transplants.
Just twenty years ago, in thirty years, admittedly with the advent of a cure for Hap C, it's now leapfrogged into the lead candidate for liver transplant. And yet, what are we doing for it? Not a lot. That's a lot I want to unpack, and as much as you still have time to discuss it, let's proceed in any order you see fit.
To add on to that, I just did a Zoom conference for University of Pittsburgh and they're a big liver center and one of their big problems with transplanting livers is living donors. They're limited by donors because they all have fatty liver, which they will not transplant because they don't do well. So not only is it the problem in treating it in terms of at least this most commonly that's the most common thing that they do, but that's an aside.
So what can we do about this if we can get our patients to lose weight? This of course is the best. Diet and exercise of course is the best thing and that's the first thing. I tell my patient, we really drill into them how we can really fix everything that's wrong with them through this process. And unfortunately, as you know and I know, it just doesn't work in the vast majority of our patients.
So in terms of pharmacology, my view and here again it's the liver, if I had to pick one organ to target It's the liver. As as important as muscle insulin resistance is at the very beginning, if we actually want to reverse the disease and make the biggest impact, if I had to pick one organ, it's the liver. If you're gonna target probably the easiest organ to target, The way I think about the liver is in terms of thermodynamics.
It's a thermodynamic problem. It goes back to my physics training. And it's really energy in and energy out. The whole metabolic problem with the liver is this imbalance of energy. Too much energy in relative to the ability of the hepatocyte the liver to oxidize the energy and convert it to CO two or export it. The one thing the liver is also able to do is export energy as a form of VLDL triglyceride.
If it's energy, how do we fix it? Well one way again we said diet and exercise, limit energy and that works. And that we talked about. Kit Peterson did this twenty years ago and shown many, many times. To get the patient to stay on this is challenging. Bariatric surgery works, again, limiting energy in. We just saw a nice study in the New England Journal. There's no magic to ruin white. It's simply if you pair feed individuals lose the same amount of weight.
same effect. Everything the bariatric surgery is doing, at least Ruin Y, is really through reducing through the weight loss. How can we do this pharmacologically? Well, GLP1 agonists are out there now. They're becoming very popular. Their major effect is energy intake. Our patients eat less. Because they eat less, they lose weight.
induces nausea, mild nausea. Some people get into issues with vomiting, nausea. Mom may have to cut back on the dose. But this is how the GLP one agonists, I believe, are having its major effect is weight loss. And they are what they are. They do accomplish Reversal fatty liver to some degree. They don't normalize, but it does come down in the right direction. Why do you think the GLP one agonists lead to reduced appetite?
I I just think through working through a central mechanism, all these gut peptides lead to nausea, vomiting, glucagon will do it, somatastatin will do it. All these things if you give'em a high enough concentrations lead to some degree of nausea and vomiting. To me it's part of a spectrum and If you just get it right, you just get people less interested in food and they eat less. Metformin, that's the one agent we have that lowers glucanogenesis. I was just
come back it's not complex one. I I wanna challenge you on that. We can talk about that. But to me it's complex one inhibition happens at millimolar concentrations, clinically not relevant. Our concentrations of metformin and humans a metaphor are about fifty micromolar, forty to fifty micromolar, not millimolars, which is what inhibits complex one. And I think it's downstream. It does affect the mitochondria, does lead to the redox, but it's not through the complex one. It's probably
indirectly inhibiting mitochondrial glycerol phosphate dehydrogenase. That's what leads to the redox. But we can come back to that if you want. I'd love to. That's very interesting. To focus then on other mechanisms, so GLP1, limit food intake. Energy expenditure, SGLT two inhibitors. Cause glucose loss in the urine, 400 calories a day loss. So they lose weight. Unfortunately, it seems to plateau after several weeks.
And you get very mild reductions in liver fat, unfortunately. Not as much, but maybe in combination with other things it might be certainly helping the right direction. My favorite target is to promote mitochondrial inefficiency. And so one of the things we're working on now is to mitochondrials where you burn the fat
That's the organelle that burns the fat through oxidation. If you can promote then the mitochondria be a little bit less efficient, so they have to burn more fat to generate the same amount of ATP. This we've shown in various forms, preclinical models, mice, rats with fatty liver, nash, liver fibrosis, it reverses fatty liver through these mechanisms, reverses Nash.
reverses the insulin resistance through reductions in DAX acetyl CoA, reverses diabetes and ZDF models. For the Nash world it reverses the inflammation and liver will reverse liver fibrosis. And so I'm very excited about this because I think it can be done safely. More recently we've done this in non-human primates and showed safety and efficacy of this approach in non-human primates. So based on the mechanisms
I've described, I think it fits. And not only what I'm very gratified by is it actually reinforces the mechanisms I've described here by reversing diabetes insulin resistance by lowering DAGs and acetyl-CoA, but it's also going to be heart-healthy. And I want to emphasize this point because Many drugs we have now for NAFL and ash reduce liver fat, maybe reverse the fibrosis or slow down the fibrosis. But they may lead to alterations of cholesterol in the wrong direction. Cholesterol goes up.
And again, I have to come back to a nice point you made is it's heart disease that is killing not only our diabetic but also fatty liver patients. It's the heart disease. So whatever we're doing to reverse fixed NAFL Nash, liver fibrosis, it has to be heart healthy. And so when you burn fat in liver through this mechanism, you decrease VLDL export, you lower triglycerides, you raise HDL, and you actually have secondary beneficial effects on the periphery. So you actually will secondarily improve
muscle fat reduce muscle fat and muscle insulin resistance. So this again fits into my conceptual view of insulin resistance and would be I think a nice therapeutic approach that we're going after. Now does the uncoupling lead to Excess Ross. creation or anything else, anytime I hear of uncoupling in the mitochondria, which is a deliberately induced form of inefficiency, you wonder is this an unintended consequence potentially?
So uncoupling by definition, the biophysics of uncoupling, the energy has to go somewhere. It's dissipated as heat. You're burning more fat. Changes in the energy is going to lead to a little bit of heat production. You will get energy production in the form of heat.
but because it's liver targeted has no effect on body temperature, will not affect whole body weight. It's interesting, I can just tell the story of uncouplers. Uh your listeners might be m interested in this. So They were first discovered actually in the early nineteen hundreds in the munitions factories. Europe was getting ready, they knew a world war was coming, the munition factories were all getting geared up.
Some of the workers in the munition factories were getting this dust, yellow dust on their hands and actually Losing weight. They were just going home and despite eating the usual amount, they're finding their weight was going down and maybe they were sweating a little bit more, a little diaphoresis, and they went to their doctors and told them about the weight loss, despite eating the same and
It's a little bit more diaphragm, more sweating. And the doctors just said, What is this yellow dust on your skin? And why don't you just wear gloves, wash your hands and wear gloves? And they got better. This was dinitrophenol. This was a substance that was used in the munition factories to make TNT, so dinitrile T and T. A physician, Tainter, in the nineteen thirties, basically said maybe this is good for weight loss, actually introduced dinitrophenol as a weight loss.
drug. It was available over the counter. It wasn't a prescription. So anyone could go like buying vitamins, get some DNP for weight loss. It actually worked. So a lot of people, hundreds of thousands of people took dinitrophenol for weight loss. And it worked. The papers published in very good journals, JAMA by Tainter and others, really described its beneficial effects. Unfortunately, and a very big unfortunately is again one of the
On target effects, we just talked about, when you uncouple, you promote heat generation. And this is in the whole body. The NP is going everywhere and promoting heat generation. Unfortunately, a handful of these people took too much, they got into problems with hyperthermia, increased body temperature, and got very sick from that and some died. W the very first thing, a newly created FDA, nineteen thirty seven, the first act they did was actually to pull
DNP from the counters as an over-the-counter kind of drug or medication. And the second ACT they had actually was thalidomide, which they pulled and now is actually back in the clinic. That was always the problem with DNP, why again we say this is not a good thing, this is a toxic drug and everything else and as it is.
it occurred to us that the reason it's generating the heat is you're uncoupling all the organs in the body and what if we just picked one organ, i.e. the liver where the fat is accumulating, this is where the organ that's driving lipidemia, hyperlipidemia, and diabetes. And if we could just melt the fat away with in a liver specific manner, maybe we can have
that beneficial effect without the toxicity. And so in a series of studies, we were able to show proof of concept That by simply uncoupling the liver, you could avoid hyperthermia and all the toxicities that have typically been associated. with the parent compound DNP and increase the therapeutic window. Every drug has a therapeutic window, even aspirin and tylenol, by a hundredfold.
Based on this thinking, I think it can be done very safely and via treatment for very important metabolic diseases like naphyll and So the IND has already been filed for this. Is it in phase one human yet? No, no. We're still exploring preclinical models, thinking potentially about
first starting out where there are no indications for things like lipodystrophy where leptin's not working. So I think my thinking is I'd like to go slowly here. Hopefully with the next year or two w we may be in humans. I think initially going after orphan diseases where there simply is no other treatment. And that would be certain forms of lipodystrophy where they get very bad diabetes, NAFL NASH, and especially in conditions where leptin's not working.
Jerry, this has been obviously as I said a pretty technical discussion, even by the standards of our podcast. I think the show notes are gonna be integral because your figures I think frankly are very helpful. As I said, I understand this content probably better than most, and yet I still find it very helpful to be able to kind of go through schematics. So gonna encourage the listeners to do that. You also have some fantastic lectures online.
I think for the people who really want to go deep into this stuff, I think frankly some of your review articles and some of your recent publications are just a a great place to go. As I said at the outset, I just think that this is the nexus from which all diseases come. stand. And therefore we are really making a mistake if we want to treat chronic diseases in their silos and just think about atherosclerosis and just think about cancer and just think about Alzheimer's disease.
without understanding how these diseases are fed. And unfortunately, that means rolling up our sleeves and understanding insulin resistance. There's simply no getting around this. If this topic were easy, you would have presented it in an easier manner. It's not easy. If I were to just kind of leave you with sort of We've talked about exercise, we've talked about nutrition. Do you feel strongly about
any form of dietary thinking. So for example, I have found clinically that carbohydrate restriction is a very effective way for patients with insulin resistance to lose weight. Not uniformly, but it's quite effective. It also seems to be easier to adhere to than outright caloric restriction, though periodic fasting also seems to do a good job. But have you observed anything similarly from a clinical perspective?
that fructose restriction specifically or sugar restriction specifically as a vehicle to weight loss becomes a more effective tool to ultimately produce what's understood to be efficacious, just some reduction of weight, either as the cause or effect of the improvement. My thinking here is what I tell my patients is whatever works.
to everyone is so different, different likes, different dislikes. I say look at the scale, whatever works for you to lose weight, because I know if you lose the weight, your diabetes is gonna get better. So I say you find something
whatever works for you, stick with it. That's the challenge'cause we're very successful in the short term getting patients to lose weight. The unfortunate part is they're able to get the weight off and then three months later, six months later, they come back to the office and they're right back where they started. So it's a matter of I tell them you have to find something that works for you, get the weight off, but then you have to be able to stick to it.
And that's where the challenge. A lot of diets, people are able to get on, they get the weight off, and they just can't adhere to it for the long term. And so it's a marathon. You have to find something you like, like it enough to be able to stick with that. That's the most important thing because We've all seen that where people lose the weight and then a few weeks, months later, right back to where they started. So everyone has to find what works for them.
I guess I want to come back to the Metformin thing because it's so interesting. So you mentioned that the inhibition of complex one actually is probably not taking place because you actually mentioned basically a thousandfold difference in concentration. Say a little bit more about that and why you're then imputing that it's the impact of metformin presumably on NAD and NADH, which you could also get out of an inhibition of complex one, but via some other mechanism, it sounds like.
Studies that we've done and we're still working on this, clearly most of the literature if you read on metformin let's talk about the big picture. So metformin lowers glucose. in patients with poorly controlled diabetes. Mostly through inhibition of glucanogenesis. I think most clinical physiologists would agree with that. And so we've done studies quantifying glucogenesis both by NMR, heavy water, multiple methods, same individuals.
And that's its major effect, not through inhibition of glycogenolysis, not through gut biome, it's gluconeogenesis. And the other thing clinically is the more poorly controlled diabetes, the greater the effect. You're not going to see much effect.
There's very confusing studies that have been published in non-diabetic individuals that find all kinds of other things going on. I don't think that's clinically relevant. It's glucanogenesis. So then how does it do glucanogenesis? So most of the literature, if you read it.
virtually all enamels that study mechanism have implicated complex one. And we've known about guanide inhibition. Metformin is a guanide, big guanide. Even before metformin we had fenformin and other guanides that have been studied. and they will inhibit complex one, no doubt about it. So and most have focused on complex one inhibition.
leading to either AMPK activation or buildup of a metabolite that inhibits glucanogenesis or something, ninety-nine percent of the mechanisms have talked about complex one inhibition. My issue with that is again uh not very many studies have done careful measurements of this
most commonly used drug on the planet. For your readership, guanides have been used for diabetes for hundreds of years. The French lilac extracts have been used 300 years ago been description. They didn't know what diabetes was at that time, it wasn't defined, but patients with polyuria, polydipsia, who are overweight, treated with the extract, the French lilac, and their symptoms improved.
Most studies if you look at or used at millimolar concentrations and again when they look at complex one inhibition, which has been implicated to then lower ATP, raise ADP, and activate AMPK. It requires millimolar concentrations. And so when you actually measure metformin in the patient who's taking one gram twice a day, which is your maximal dose and pretty much the best efficacious dose.
Your levels in plasma are about thirty to fifty micromolar. So you could say even, you know, in portal vein its pills are taken orally, you give it two to three times that. You're still talking about maybe a hundred micromolar. tenfold less than what all of these studies have been doing, even both the in vitro studies in the literature and well as the in vivo studies giving levels that achieve millimolar concentration.
So yes, you see things. Complex one is an important, it's an electron transporter, it's important for function and health, and you're going to see effects when you inhibit complex one at those high concentrations. In my view, they're not clinically relevant. So the effects that w I do think are clinically relevant that we have observed. at fifty and a hundred micromolar of metformin are really on the enzyme glycerol three phosphate dehydrogenase. The mitochondrial isoform
That is required to move the protons from outside to inside the mitochondria. And when you inhibit this enzyme, NADH goes up, NAD goes down. When you have this increase in the cytosolic redox, you can't get lactate to pyruvate and you can't get glycerol to DHAP. So if I'm right, it's going to be substrate dependent. inhibition of glucanegenesis. Whereas if you inhibit complex one and AMPK or whatever mechanism downstream, it should be glucanegenesis independent of substrate.
And what we've shown both in vitro and in vivo, most importantly in vivo, in two or three different models. Metformin at these clinically relevant doses and concentrations only inhibit glucanogenesis from glycerol. And lactate. It doesn't inhibit it from alanine or DHAP or anything that does not depend on the cytosolic redox state. This also explains why we rarely see clinically hypoglycemia on patients treated with metformin, because there's these alternative
Glucanogenic substrates that can come in alanine can keep coming out. So you never see, rarely unless they have another agent on top of metformin like insulin or SU, you rarely see it, if ever. And that's why also you see the lactic acidosis. Which is a fortunate toxicity of metformin where again it's s specifically getting that lactate to pyruvate conversion, which is dependent on the redox date. So
That's the mechanism I believe is clinically relevant. And now we're the last step is how is it inhibiting this enzyme? And I believe it's actually through an indirect effect on this enzyme that we'll hopefully have ready for prime time in in the year. And do you think that in a healthy individual who's eating well, is of normal weight, is insulin sensitive, and is exercising robustly? Metformin could actually be counteractive to benefit.
That's a r profound question. I don't know the answer to that. And it gets into I don't know if you're gonna take me there, in terms of the use of metformin for aging. Healthy people are taking it for aging now. I think that's why it's so important to understand this mechanism, then understand the implications of if it is redox. Is that a good thing or not for longevity and health?
That's a question that remains to be answered. I find myself very much on the fence with that question while in the insulin resistant patient, even without diabetes feeling that this is a very net positive agent, but my personal views on it just based on clinical observation is that in the person I described earlier, the lean, insulin sensitive, vigorously exercising individual, it may actually not provide benefit. But
Again, there are studies in the works that are gonna hopefully be able to provide some fidelity to understanding that. I it sounds like you're equally kind of undecided on that as well. Yes.
Well, Jerry, I can't thank you enough. Again, I say this to many people I interview, but I really mean it here. It's not just for this discussion and the time you put into it, but obviously much more importantly for the career and for this incredible body of work that you've amassed through your pursuit and obviously remarkable collaborations with so many people. I've enjoyed this discussion immensely.
It's actually one of the discussions I'm gonna have to probably go back and listen to again. So I hope that a listener isn't hearing this and isn't discouraged by the fact that you're at this point in the discussion and you're thinking, Oh my god, I might have only retained half of that. That's okay. I'm gonna be listening to this one and I just finished listening to it now and I'm going to listen to it again. So thank you very much, Jerry, for that.
Obrigado, Peter. Foi um prazer. Thank you for listening to this week's episode of The Drive. If you're interested in diving deeper into any topics we discuss, we've created a membership program that allows us to bring you more in-depth exclusive content without relying on paid ads. It's our goal to ensure members get back much more than the price of the subscription.
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