The Peter Attia Drive - #140 - Gerald Shulman, M.D., Ph.D.: A masterclass on insulin resistance—molecular mechanisms and clinical implications

Episode Date: December 7, 2020

Gerald Shulman is a Professor of Medicine, Cellular & Molecular Physiology, and the Director of the Diabetes Research Center at Yale. His pioneering work on the use of advanced technologies to analyze metabolic flux within cells has greatly contributed to the understanding of insulin resistance and type 2 diabetes. In this episode, Gerald clarifies what insulin resistance means as it relates to the muscle and the liver, and the evolutionary reason for its existence. He goes into depth on mechanisms that lead to and resolve insulin resistance, like the role of diet, exercise, and pharmacological agents. As a bonus, Gerald concludes with insights into Metformin’s mechanism of action and its suitability as a longevity agent. We discuss: Gerald’s background and interest in metabolism and insulin resistance (4:30); Insulin resistance as a root cause of chronic disease (8:30); How Gerald uses NMR to see inside cells (12:00); Defining and diagnosing insulin resistance and type 2 diabetes (19:15); The role of lipids in insulin resistance (31:15); Confirmation of glucose transport as the root problem in lipid-induced insulin resistance (40:15); The role of exercise in protecting against insulin resistance and fatty liver (50:00); Insulin resistance in the liver (1:07:00); The evolutionary explanation for insulin resistance—an important tool for surviving starvation (1:17:15); The critical role of gluconeogenesis, and how it’s regulated by insulin (1:22:30); Inflammation and body fat as contributing factors to insulin resistance (1:32:15); Treatment approaches for fatty liver and insulin resistance, and an exciting new pharmacological approach (1:41:15); Metformin’s mechanism of action and its suitability as a longevity agent (1:58:15); More. Learn more: https://peterattiamd.com/ Show notes page for this episode:  https://peterattiamd.com/geraldshulman  Subscribe to receive exclusive subscriber-only content: https://peterattiamd.com/subscribe/ Sign up to receive Peter's email newsletter: https://peterattiamd.com/newsletter/ Connect with Peter on Facebook | Twitter | Instagram.

Hey everyone, welcome to the drive podcast. I'm your host, Peter Attia. This podcast, my website, and my weekly newsletter all focus on the goal of translating the science of longevity into something accessible for everyone. Our goal is to provide the best content in health and wellness, full stop. And we've assembled a great team of analysts to make this happen. If you enjoy this podcast, we've created a membership program that brings you far more in-depth content if you want to take your knowledge of this space to the next level. At the end of this episode, I'll explain what those benefits are, or if you want to learn more now, head over to peteratiamd.com forward slash subscribe.

Now, without further delay, here's today's episode. My guest this week is Dr. Gerald Shulman. Jerry's a professor of medicine and cellular and molecular physiology at Yale. He's also the co-director of the Yale Diabetes Research Center. In 2018, he received the Banting Medal for Scientific Achievement, arguably the most prestigious award one can win in this field. He's pioneered the use of magnetic resonance spectroscopy combined with mass spec to non-invasively examine cellular glucose and fat metabolism. Now, you may ask, why does that matter? And we get to that right at the outset of this interview. If you want to understand insulin

resistance, if you want to understand hyperinsulinemia, type 2 diabetes, non-alcoholic fatty liver disease, you have to understand the movement of glucose and fat. A way to think about this is to think about it this way. If you go and get a blood test, even the most fancy blood test imaginable. You're basically looking at a picture, a snapshot, a moment in time. What these techniques that Jerry and his collaborators have developed over many years are basically allowing you to watch videos. You can see the flux. You can see the movement of glucose. Furthermore, they've been able to see those things as they happen inside even human cells. So I'm going to, I guess, maybe make an apology at the outset of this and say that this is about as technical an interview as I probably have done in a while.

There are probably only a few interviews that I've done on this podcast that get into more technical weeds than this one. But unfortunately, that is the price one has to pay if they want to understand arguably the most important pathologic condition in our species. And I get into what I mean by that in the interview, so I won't elaborate on that further. We talk a lot about what it is to be insulin resistant. This term gets thrown around with such ubiquity that you'd think everybody knows what it means, and yet to define it is very complicated. But I think by the end of this interview, you will undoubtedly understand how to define

insulin resistance. And I think you will have a very good sense of where it begins and what it means in a subclinical versus a clinical state and what the sequence of events are that lead to a condition in the muscle, ultimately affecting the liver, ultimately affecting the whole body. I'm not going to promise you that you'll get that on the first listen. I think that might require more than one listen, and it probably requires going through the show notes, which will be littered with fantastic figures. In fact, as Jerry and I did this interview over Zoom for, I don't know, I would say maybe half to two-thirds of the interview, he was sharing slides as we were doing it. And keep in mind, I'm quite familiar with these concepts. So for someone who's less familiar with these concepts, I think it will be only that much more valuable. One really nice little bonus thing that came out of this is a beautiful discussion at the end of Metformin where I

actually learned something really profound that was incredibly relevant to my understanding of Metformin in my ongoing interest around the question of Metformin's suitability as a longevity agent. I could say more about this, but I think it's just one of those things where I ask you to sort of take a leap of faith with me that this is important, even if it feels a little bit like drinking your cough syrup at times. But if you really want to understand longevity, you're going to have to sort of figure out what insulin resistance means. So without further delay, please enjoy my very in-depth conversation with Dr. Gerald Shulman. 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 guess I'd like to kind of just start with a little bit of background. You did an MD and a PhD and you're trained as an endocrinologist, correct? Yeah, that's correct. And then I did residency in medicine at Duke, a fellowship in endocrinologist, correct? Yeah, that's correct. And then I did residency in medicine at Duke, 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 diabetics. At an early age, I was exposed to problems, type 1 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 because my physics background, but I ended up staying in metabolism 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 as an undergraduate medical school, I was always interested in biochemistry, physiology. I had an experience, I was visiting a medical student at Vanderbilt in the 70s and got interested in in vivo metabolism, doing study 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 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. The cost to U.S. 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 just setting up methods, this technique to actually look inside living yeast cells. I said, gosh, this we can adapt this to 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 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 2 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 2 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 2 diabetes, but it also, and again, this is give credit to Jerry Riven, who in his 1988 Banting 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 fatty liver disease, MAFLD. That, or I prefer the term metabolic associated fatty liver disease, NAFLD. That's 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 budding 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 cancers, which are associated with obesity, breast cancer, colon cancer, pancreatic cancer, liver cancer. And by the bed, 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. 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 in insulin pumps. It's simply, and that 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 2 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, more than 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 50 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 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, glucose. And so you're used to basically, with the advent of cyclotrons,

it really started in California. In Berkeley, they started, you know, had cyclotrons, they're 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 50 years, we've been able to buy radio labeled isotopes and put a carbon, 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 us 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. And 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 cell. 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. 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 were two groups that were pioneering this kind of work. There was one group in George Rada at Oxford, and this was phosphorus NMR. 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 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, 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, C13 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 C12, which is NMR invisible, but 1% is C13, which is NMR visible, has this precession properties. You can use a labeled, for example, C1 labeled glucose and then track that C1 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 NMR, as getting back to George Roddick, George pioneered phosphorus NMR. There you don't have to give any isotopes. There you actually see P31, phosphorus 31 is 100% 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, all the high energy phosphates in, for example, ATP, adenosine triphosphate. There are three phosphates. And you can actually see each one of those phosphates. You can see phosphocreatine has a different chemical shift. You can see inorganic phosphate. And we develop methods, Doug Rothman

and others at Yale who I work with, we're able to develop methods to measure glucose 6-phosphate. So we can actually look at 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 an 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 carbohydrate, how much of that carbohydrate ends up in glycogen versus

oxidation into carbon dioxide or gets converted to lactate through glycolysis? 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? This story is very short. glucose to glycogen accounting for their insulin resistance. This story is very short. Before you go there, let's demonstrate clinically a difference between these two people. So let's take the normal patient without type 2 diabetes, and then let's contrast them with a very similar person of similar size who has type 2 diabetes. We will feed them both a high-carbohydrate meal in the evening. Let's just assume that that meal contains 100 to 200 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 2 diabetes might arrive with a blood sugar of 100 milligrams per deciliter, which we will say is normal. His counterpart with type 2 diabetes may actually at that time have a blood sugar of 200 milligrams per deciliter, which is obviously abnormal and consistent with the diagnosis of type 2 diabetes. Now, of course, that only represents about an extra five grams of glucose in the circulation that is the difference between the 100 and the 200 milligrams per deciliter, which is a small fraction of the, call it one to 200 milligrams,

pardon me, grams rather, five gram difference. So it's a small fraction of what, call it one to 200 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 depends on the size of the meal and how it's being administered, the proportionality between liver and muscle. But the meal and how it's being administered, the proportionality

between liver and muscle. But the bottom line, it's 80, 90% 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 gluconeogenesis, the conversion of non-glucose precursors like amino acids, alanine, and lactate to glucose. And that process is accelerated. So the liver is 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 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 is 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 2 diabetics and short-term hypocaloric feeding, 1,200 calories a

day. We basically can reverse all these abnormalities through reduction in ectopic 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, Rory Taylor, my colleague who trained with us, is now doing this in the primary care clinic back in the UK. But usually, you've asked the question, usually when we talk about diabetes, actually, it may be easier to understand when you start in the young, lean 20-year-old who already has insulin 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 is the most common thing. As I said, probably half the people in the U.S. 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 2 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, and we challenge them with an oral glycemic load and then measure insulin and glucose in 30-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 say 90, you challenge them with 75 to a hundred grams of glucose, but say 30 or 60 minutes later, their fasting glucose is 200. Their insulin is 70. 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 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 is 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 going to be the root cause for all these other abnormalities.

Fatty liver disease makes 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 going to make a big impact there, fatty liver disease, and slow down cancers. 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 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 it puts the breakdown of fat, and it's called atlipolysis, 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, keep emphasizing, I think for your listeners, this is probably every other person in this country or 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 the 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 20 years of age, 19 to 20. 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 22, 23 BMI, lean. Non-smoking, so we screen out smoking. No medication, 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 20-year-olds, let's say you screen, we screen to this date probably 1,000, 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, 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 in the 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 probably 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, wherever that roadblock is, where the construction is 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. 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 these intermediates. 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 at transport, there should be reductions in both

glucose 6-phosphate and glucose. Through 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, implicating transport that's where your biochemical block is so the block is at transport that's your target to fix if you want to fix muscle insulin 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?

That led us into the world of lipids. Again, it's been known for decades that obesity is 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 the splock 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, IRS1, inside the cell. So that sends a signal inside the cell, which also leads to a phosphorylation, which then signals PI3 kinase. It upregulates PI3 kinase. And that basically leads to a glute

four 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-1, with PI3K? Is there something blocking the transporter? We're going to 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 going to take everything that you are sharing with me and we're going to turn these into show notes that will be timestamped 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 will 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 to the receptor,

the receptor autophosphorylates, becomes a kinase. The key substrate for this kinase, this receptor kinase in muscle is insulin receptor substrate 1, which undergoes tyrosine phosphorylation, allows it to bind and activate this 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 going to 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

are obese and insulin resistant, or even these young, lean, insulin-resistant offspring. And so this is the picture. So here, and it relates to fatty 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. This is, people go into a scanner and they get very pretty pictures of an organ brain or some other organ for diagnostic reasons. And it's the same biophysical principles. 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 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 NMR 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. 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 2 diabetes. And others had shown that in addition to us. I mean, 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 diacylglycerol? No, this is a triglyceride. This is an emulsion, a triglyceride emulsion. It's often given to patients for hyperalimentation when they can't eat. You give this energy-rich infusion. Just like TPN or something like that. It's TPN. 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 up to about 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 going to get more of that lipid into the muscle cell. You will raise fatty acids. 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 deliver fatty acids to all cells in the body. Yeah. 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 giving

fatty acids. Fatty acid turnover is so fast. You can't just infuse fatty acids to 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 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, 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 resistance, as I say, as anyone with obesity or type 2 diabetes. And it's due to the same, an acquired block in transport, insulin activation of transport, both G6P 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, who did some pioneering studies in the 60s at University of Bristol, and was really the first to say, hey, fatty acids may be toxic, may be 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 because 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 postulate mechanism, translates to humans, because these were all in vitro studies done in tissues taken from animals. So in a series of studies, we took, again, the 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 G6P 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's 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 pre-diabetic 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 going to now fast forward. We then took these observations back to the bench. We're able to replicate this in rodents, rats, and mice.

And the power, even though I'm most passionate about our human studies, I'm a clinical physiologist, and I care most about understanding what's happening in humans. The animal models allow you to really interrogate 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 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 fatty acid metabolite interfering with insulin signaling cascade, which we just discussed, receptor and somewhere to PI3 kinase. which we just discussed, receptor and somewhere to PI3 kinase. And what we found was indeed in healthy individuals, just give glucose and insulin, you get activation of PI3 kinase. This is the step you mentioned. This is the required step for translocation. And in the follow-up study, same individuals, we raise glucose and insulin and also raise fatty acids. we raise glucose and insulin and also raise fatty acids. And then we totally abrogate insulin activation of PI3 kinase. 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 focused lipidomics, we zeroed in on this metabolite, fatty acid metabolite called diacylglycerol. And yeah, I heard you mention that before. It's the precursor, it's the penultimate step in triglyceride synthesis, diacyl-2 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 trapped with insulin resistance. And then we did the lipid, same type of lipid infusion we did in humans, simply raise plasma fatty acids by giving triglyceride and heparin. We saw acyl-CoAs go up. We saw DAGs go up. Right when DAGs reached a peak, then we got activation of novel PKCs, PKC theta and epsilon in the muscle. Then we link to this block in 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 going to do the same thing if we have time to talk about the liver, because that's the other key insulin response of Oregon. But we'll start with muscle. Fatty acids are being delivered either through fatty acids or even hydrolysis of triglycerides through LPL, etheliobaric, 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, acyl-CoA is triglyceride, you get net accumulation of diacylglycerol. This is a very important point. Triglycerides are neutral. So I want to 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 an inert 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 triacylglyceride or triglyceride, those two we use interchangeably, has this three carbon glycerol backbone with three free fatty acids on it. 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 TG or TAG? 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 neutral lipid, 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 IRS1 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 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 going to 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 PI3 kinase activation, and as we talked about, then less GL 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 npkc theta you get protection we've been able to block mito oxidation and you make these animals prone to that build up 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 20 year old, basically undergrads at Yale that we're going to 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

Riven 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, and I want to do two things. I want to show you how exercise reverses this muscle insulin resistance. But I also want to 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 Reuben's Banting Lecture in 1988. 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 2 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 20-year-olds, these volunteers, is the first thing we see is muscle insulin resistance, and maybe that's driving atherogenic dyslipidemia, who is 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 healthy, young, sedentary individuals, we're going to 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. muscle, as well as glycogen in liver and muscle. And what we found then is you give them two high carbohydrate milkshakes, and there's virtually no difference in the plasma glucose concentrations at this late breakfast and lunchtime high carbohydrate shake. But you can see it's at the expense of severe hyperinsulinemia is what we talked about. So 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 new glycemia. 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 insulin resistant person has normal glycemia because the beta cells are working so hard to maintain this. And you can see that here.

The other thing I want to point out is the insulin levels are given 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 with the liver sees is three times this. Liver seeing huge amounts of insulin in these insulin-resistant individuals just to maintain normal glycemia. We use carbon NMR 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 fat and this carbohydrate, this is change in liver triglyceride, it's up two and a half to 0.3 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 94-ish. And he looked at this question of how much carbohydrate could be converted to fat via de novo lipogenesis. 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, which suggests that an insulin-resistant person is capable of significant de novo lipogenesis. Everything you've said is correct. When you're thinking about de novo lipogenesis, two things is, again, what conditions are you studying this? Is it after meal ingestion? Is it in a fasting state when a lot of people have measured this in the past? It's minimal, and it makes sense.

It only gears up with 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 esterification, that is fatty acids coming to the liver, getting incorporated into 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 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 2007 PNAS 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 post-prandially, nevermind 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 150 milligrams per deciliter is considered normal. Well, we don't say that. In our practice,

we view anything over 100 as abnormal. That's a red flag. And if your trigs are more than 2X 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 novo lipogenesis, and threefold difference we see in de novo lipogenesis, hepatic synthesis of fat, impaired hepatic glucose sensitivity. 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 and something we're quite passionate about. And I want to kind of 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 per if it's up to five, 600 microunits per mil. That turns on SRIBP1C, 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 going to set these healthy individuals up to atherogenic dyslipidemia, heart disease in their 40s and 50s. 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 going to be liver cancer. So it's all going to 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 Joslin group did a really nice study. They found that if you have two parents with type 2 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 2 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 just studied them in the basal state, shown here that, you know, again, he's young, insulin resistant. Again, everyone here is lean, non-smoking, no medications. They're in their 20s and 30s, BMI 23, 24 to factor out obesity, confounding the factors of obesity, medication, smoking, other things. So young, lean, healthy individuals, but just parents with diabetes, insulin resistant. 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 being on a Stairmaster, three 15-minute bouts at about 65% MbO2 max. And here we're normalizing insulin-stimulated muscle glycogen synthesis. And we're 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 AMPK, which we can talk about, gets activated with exercise. And that has been shown to cause borer translocation independent, independent of PI3 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, 45-minute bout, was sufficient to open up the door to glucose, cause that GLUT4 translocation. And Rasmus Röbal, when he was a clinical fellow with me, did one single bout in these same individuals I showed you before, insulin-resistant in muscle, the ones had high triglycerides, low HDL, and prone to increased TNL. With a single bout, we were 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? Where was the actual bottleneck alleviated with that single bout of exercise? I can speculate. In these human studies, I can tell you that we open up the door, we measure glucose 6-phosphate in them, and that 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're getting improved insulin signaling at the level of the receptor and IRS-1, and or is it just AMPK causing this GLUT4 translocation? If I had to speculate, I would think most of it is through the latter. We were simply with an acute bout causing AMPK-induced GLUT4 for translocation which we know happens independent of pediatric kinase that's established so we're short-circuiting we're just causing glute 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 glucose 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 could walk around with a hemoglobin A1C of 6% 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, 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 want to understand this. Yeah. That's kind of fun. So let's now turn. So I 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 dyslipidemia, 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 insulin resistance, 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 in different stages. We don't see liver abnormalities in these young 20-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 the reverse direction. They get liver fat first, liver insulin resistance, and then muscle. Most of the studies 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 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 and postprandial. Here's the cartoon of the liver cell. And again, glucose transport is not rate 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, use piyothrekinase, 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 gluconeogenesis through a transcriptional regulator. And that then is excluded from the nucleus and then down regulates then gluconeogenesis 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 is inhibiting this key process. So let's now just focus on how lipid causes insulin resistance in liver. Same metabolite, it's the diisoglycerols. They go to activate epsilon. That's really the major isoform of PKC, novel PKCs in liver. And work by Varmin Samuel, when he was doing his PhD with me in a series of studies,

Varmin 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, 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 Reinhardt 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-1, IRS-2 can't go into the pocket for tyrosine phosphorylation. When insulin binds the receptor, these three tyrosines, the 1158, the 1162, and the 1163 become phosphorylated, that opens the door, that loop flips out, and then IRS1, IRS2 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 in MassSpecMaven, identified using purified receptor, purified PKC epsilon, that when you add activated epsilon to the receptor, you phosphorylate this threonine. And that got us very excited because, golly, that's one amino acid away from these two

tyrosines that are required for activation receptors. Maybe 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 threonine with glutamic acid, mimic a some genetics. Again, that's what you can do is you can knock a glutamic acid, replace that threonine with glutamic acid, mimic a phosphorylation event, and that kills the kinase activity. You can mutate the threonine 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 11, this is the mouse homologue, the 1150 is the homologue for the 1116 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 insulin

sensitivity, nothing, normal size, normal growth. But when Max 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 days, they get 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 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 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-nutrient alanine, 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. 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, of diisoglycerol, and it really matters which isoform it is and 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 diisoglycerols, it's really the SN1-2 isoform, 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 SN1,2 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 showed both that PKC epsilon is both necessary and sufficient for this process by doing the knock-in and overexpression. But I just want 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 lipolysis, more fatty acids delivered to the liver, hepatic fat accumulation, DAGs we show go up, SN1,2, 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 CNS,

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 threonine 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 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 cave fish found this cave fish 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 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 want to make sure I can unpack

this a little bit. So I want to make sure I can unpack this a little bit. So I want to start in the muscle because 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 48 hours of fasting in a mouse is a seismic fast, a near fatal fast. But let's say 48 to 72 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 40 days of starvation, humans were still getting about 40% 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 focus 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 the threonine 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 20 years ago when I was first studying it, maybe 30 years ago. Insulin binds the 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 gluconeogenesis. 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 gluconeogenesis, this process that

is required to keep us alive during starvation. Without gluconeogenesis, we're not going to wake up in the morning because it's gluconeogenesis 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 10 minutes without gluconeogenesis as a species. Well, I'll modify that a little bit.

As passionate, I'd love to hear you state the importance of gluconeogenesis. No, we know clinically you can. And again, from the lessons learned from Gene Knockout, you know, unfortunately, there are patients with inherited disease, von Gehrke's disease. As you know, patients who don't have glucose 6-phosphatase, the last key step getting glucose 6-phosphate out. We do know that can be compatible with life. We have patients with glucose 6-phosphatase, 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 gluconeogenesis. Otherwise, glucose would go up to 400 or 500 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 drives fast in hyperglycemia. Traditionally, pretty much the major textbooks, physiology, biochemistry,

insulin is thought to be turned off gluconeogenesis through transcriptional mechanisms. And again, this is this FOXO phosphorylation by AKT, exclusion from the nucleus. Then you get downregulation of PEPCK, excuse me, and 6-phosphatase. FOXO is the transcription regulator for these downregulation. The problem with this view, and again, there's some beautiful molecular biology, And I don't want to deny 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 gluconeogenesis in a fasted mouse, which is totally dependent on gluconeogenesis. That speaks to

the fact you don't need these key insulin signaling pathways to regulate gluconeogenesis. 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 Roux-en-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 so we can do actually protein measurements and enzyme measurements of Pepsi K6 phosphates. 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 when I was a luxury medical student,

I expected Pepsi K and 6-phosphatase and fructose 1, 6-biphosphatase all to be upregulated two to threefold in the poorly controlled diabetic that was undergoing through and bypass surgery compared to the non-diabetic. And we found no relationship between protein expression of these enzymes, gluconeogenic 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 gluconeogenesis, 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 gluconeogenesis within five minutes, and that's much faster than

you'd expect in transcriptional translational mechanisms. Just to kind of talk about how gluconeogenesis, this is the gluconeogenic 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 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 gluconeogenesis. And this, in a series of studies that Anila 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 gluconeogenesis by acetyl-CoA. This had been known for decades to be a regulator of pyruvate carboxylase 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 the diabetic situation. We said, well, wait a minute, let's go back and look at acetyl-CoA. We developed the methods, tandem mass 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 of beta-oxidation. Take fatty acids and break them down through beta-oxidation, the end product is 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 acetyl-CoA. This explains how insulin acutely suppresses gluconeogenesis. When diabetic models, when you have increased gluconeogenesis, it's twofold increases in aceto-CoA, but it perfectly follows rates of gluconeogenesis, which we quantify, track perfectly with concentrations of hepatic aceto-CoA 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 gluconeogenesis 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 pyruvate carboxylase activity. And again, there's about 10, 15% of this gluconeogenesis 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 liver. Gluconeogenesis is mostly 90%, I would say. There may be a little

bit of intrapatic lipolysis regulation, but mostly through its effect to put the brake on peripheral lipolysis. And this model, by the way, will explain, in my view, 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 works. You do things to the periphery fat cell and you affect glucose metabolism, gluconeogenesis. 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. Mice, and as you pointed this out, Peter, even after an overnight fast, boom, all the glycogen's gone. Very different from humans. Humans hold on to their glycogen like dogs, probably for two days. We've done these measurements with starvation in humans. We've shown it. 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 24 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 and 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, so 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 brake 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 IL-6. 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 10, 20 years ago, everyone was discussing inflammation circulating cytokines, TNF-alpha IL-6 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 to fasting hyperglycemia depends on inflammation. And it's through this mechanism where now you have localized inflammation in the fat cell. TNF-alpha, IL-6, 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 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 this, and that's where acetyl-CoA goes up. And again, now your rates of lipolysis, when you measure turnover, not just fatty acid concentrations, but turnover, palmitate turnover production and glycerol turnover it's up twofold this increases acetyl coa concentrations twofold this activates pyruvic carboxylase activity and flux twofold and then in addition your glycerol delivery to liver is up twofold and now your rates of gluconeogenesis are increased twofold and this is now what's driving fasting hyperglycemia in every poorly controlled type 2 diabetes. It's this gluconeogenic process that we've shown using many, many methods, and others have shown this too. This is what now is driving hyperglycemia in type 2 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 under conditions, the liver fat is playing a role, but most of it, in most of, you know, I would say garden variety, what I see is going to be peripheral lipolysis. So when we think about an insulin resistant obese person with metabolic syndrome, so this is what, 20% 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 esterified 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, my 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 threefold. So again, their curve is right shifted. Insulin is 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 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 diacylglycerol epsilon pathway is also accounting for this defect in insulin action in the fat cell. So it's going to actually be a common mediator. And again, 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 of the fat cells. And we're seeing the same thing. And we see those same mice that I showed you before, the IRK knockin mice are protected

from lipid-induced fat insulin resistance. On the fat topic, we've talked a lot about the intramyocellular lipid-induced fat insulin resistance. On the fat topic, we've talked a lot about the intramyocellular 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 T1- 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 core set 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 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, sub-Q, if you're going to store fat somewhere,

that's the best place to store it. You certainly don't want to keep it inside liver and muscle cells. In my view, and again, studies have been done to look at the visceral fat, 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 anytime when you're doing your imaging, if you just look at the liver too, they're going to correlate one to one, 99 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 acid delivery portal vein is probably going to lead to increased acetyl vein, presumably, and again, fatty acid delivery

portal vein is 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 hepatocyte, more importantly, this 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, chock full of fat and liver, and again, diabetes through these mechanisms, acetyl-CoA driving gluconeogenesis. And that's independent of visceral fat. So that shows again, diabetes through these mechanisms, acetyl-CoA driving gluconeogenesis.

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 acetyl-CoA driving pyruvate carboxylase. And again, the diisoglycerols activating Epsilon. And again, it's the Epsilon that drives insulin resistance, no diabetes, no hyperglycemia. Then it's this accelerated gluconeogenesis 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 2 diabetes, put them on 1,200 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 and definitely we can discuss. definitely we can discuss. And of course, we've already established where these DAGs are coming from. Again, I want to pause for a moment on that because I think a listener of this right now is going to say, guys, you've lost me, okay? They don't know the difference between PEPCK, 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 sedentary 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 an R, environmental triggers contributing to these epidemics, which largely began here in the United States, but we have fabulously spread to the West 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 10 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 2% of liver transplants. Just 20 years ago, in 30 years, admittedly, with the advent of a cure for hep 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 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 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 going to 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 CO2 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 in, that works. And that we talked about, Kit Peterson did this 20 years ago and it's 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 Roux-en-Y. It's simply if you pair-feed

individuals, lose same amount of weight, same effect. Everything the bariatric surgery is doing, at least Roux-en-Y, is really through reducing through the weight loss. How can we do this pharmacologically? Well, GLP-1 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, you have to cut back on the dose. But this is how the GLP-1 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-1 agonists lead to reduced appetite? I just think through working through a central mechanism, all these gut peptides lead to nausea, vomiting. Glucagon will do it. Somatostatin will do it. All these things, if you give them 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 gluconeogenesis. I would just come back. It's not complex one. I want to 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 in humans, metformin are about 50 micromolar, 40 to 50 micromolar, not millimolar, 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 GLP-1, limit food intake, energy expenditure, SGLT-2 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 that 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 mitochondria

is 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 and NASH, liver fibrosis. It reverses fatty liver through these mechanisms, reverses NASH, reverses the insulin resistance through reductions in DAGs, acetyl-CoA, reverses diabetes and ZDF models. For the NASH world, it reverses the inflammation and 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 NAFLA and NASH 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. 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 ROS 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 and 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. Your listeners might be interested in this. So they were first discovered actually in the early 1900s 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 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 diaphoresis, 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 dinitro-TNT. A physician, Tainter, in the 1930s 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 BNP 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. DNP 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. With the very first thing, a newly created FDA, 1937, 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 it's 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 within 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 be a treatment for very important metabolic diseases like NAFLA and NASH.

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 is not working. So I think my thinking is I'd like to go slowly here. Hopefully within the next year or two, 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 is 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 going to 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 I'm going to 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 great place to go. As I said at the outset, I just think that this is the nexus from which all diseases 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 diseaselerosis 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, which is 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. 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 going to get better. So I say, you find something, whatever works for you,

stick with it. That's the challenge because 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, 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 gluconeogenesis. I think most clinical physiologists would agree with that. And so we've done studies quantifying gluconeogenesis, both by NMR, heavy water, multiple methods, same individuals. And that's its major effect, not through inhibition of glycogenesis, not through gut biome, it's gluconeogenesis. And that's its major effect, not through inhibition of glycogenesis, 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 gluconeogenesis. So then how does it do gluconeogenesis? So most of the literature, if you read it, virtually all in animals, that study mechanism have implicated complex one. And we've known about guanide inhibition. Metformin is a guanide, biguanide. Even before metformin, we had fenformin and other guanides that have been studied, and they will inhibit complex I, no doubt about it. And most have focused on complex I inhibition, leading to either AMPK activation or buildup of a metabolite that inhibits gluconeogenesis, or something. 99% of the mechanisms have talked about complex I inhibition.

99% of the mechanisms have talked about complex one inhibition. My issue with that is, again, 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 in description. They didn't know what diabetes was at that time. It wasn't defined, but patients with polyuria, polydipsia were overweight, treated with the extract, the French lilac, and their symptoms improved. Most studies, if you look at, were used at millimolar concentrations. And again, when they look at complex I 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, pretty much the best efficacious dose, your levels in plasma are about 30 to 50 micromolar. So you could say even, you know, in portal vein, it's pills are taken orally, give it two to three times that. You're still talking about maybe 100 micromolar, tenfold less than what all of these studies have been doing, even both the in vitro studies in the literature and well, the in vivo studies, giving levels that achieve millimolar concentrations. So yes, you see things. Complex I 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 I at those

high concentrations. In my view, they're not clinically relevant. So the effects that I do think are clinically relevant that we have observed at 50 and 100 micromolar of metformin are really on the enzyme glycerol 3-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 gluconeogenesis. Whereas if you inhibit complex one and AMPK or whatever mechanism downstream, it should be gluconeogenesis

independent of substrate. And what we've shown both in vitro and in vivo, most importantly in two or three different models, metformin at these clinically relevant doses and concentrations only inhibit gluconeogenesis 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 gluconeogenic 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 specifically getting that lactate to pyruvate conversion, which is dependent on the redox state. So that's the mechanism I believe is clinically relevant. And now 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 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 profound question. I don't know the answer to that. And it gets into, I don't know if you're going to 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 it. 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 going to hopefully be able to provide some fidelity to understanding that. 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 going to 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've only retained half of that. That's okay. I'm going to 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. Thank you, Peter. It's been a pleasure. Thank you for listening to this week's episode of The Drive. If you're interested in diving deeper

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