Pre-lecture Diabetic Ketoacidosis

By: Michael Hunter

Welcome to the diabetic ketoacidosis pre-lecture video. This video was made in order to spend more time in the classroom for multiple choice questions as well as talking about the clinical manifestations of diabetic ketoacidosis. The content of this video is mostly a review from what you have heard already in MTC. Here are the objectives for the video.

First, we are going to talk about how glucose signals for beta cells to release insulin. Then, we're going to describe how carbohydrate metabolism and lipid metabolism are changed both during the "feeding" and "fasting" states. Insulin, glucagon, and epinephrine play a critical role in integrating metabolism in different tissues. Insulin is a peptide hormone, meaning that it is made up of amino acids and synthesized, packaged, and secreted from beta cells, which are found in islets of Langerhans in the pancreas. Insulin is synthesized in the rough endoplasmic reticulum and is known as pro-insulin before it is cleaved into insulin and c-peptide in a one-to-one ratio. Exogenous insulin does not contain c-peptide. So if you were to measure insulin and c-peptide in a diabetic who is taking insulin, insulin levels would be higher and c-peptide levels would be lower. Remember that glucose is the primary regulator of insulin and needs to be transported into a beta cell in order to propagate a signal for insulin to be expelled out of the cell into the bloodstream.

So in order for insulin to be secreted, glucose needs to go through a transporter. There are many different transporters in the body and we're going to discuss three different transporters. The Glucose-1 transporter is found primarily in red blood cells and in the brain. It is insulin independent meaning that insulin is not required for glucose to go into the cell. Remember that red blood cells are solely dependent on glucose for energy whereas the brain in fasting states can use ketones for energy. Glucose-2 transporters are found in beta islet cells, liver, kidney, and the small intestine. These transporters are bidirectional, meaning that glucose can go not only into the cell, but glucose can go out of the cell.

As you may remember, liver is the primary area where gluconeogenesis occurs, but gluconeogenesis and also occur in the kidney and the small intestine. So it's important for these organs to have bidirectional glucose transporters because gluconeogenesis is occurring and that glucose needs to be secreted into the blood. Glucose-4 transporters are mainly found in adipose and skeletal muscle and these transporters are insulin dependent so they need insulin in order for glucose to go into these cells.

Pre-lecture Diabetic Ketoacidosis

Insulin must be present. One way that I remember which organs or which areas of the body have insulin independent transporters is by the acronym BRICK-L. BRICK standing for brain, R standing for red blood cells, I standing for intestine, C standing for cornea, K standing for kidney, and L for liver. Now we are going to talk about how insulin is secreted from beta cells, so I'm going to draw here a beta cell. And then I'm going to draw a glucose. In order for glucose to get into the cell, there needs to be glucose-2 transporter. Remember this is bidirectional and glucose goes into the cell and converted to glucose 6-phosphate. It's phosphorylated and this phosphorylation is done by glucokinase.

And this phosphorylation kind of traps the glucose in the cell. Glucose 6-phosphate then goes through the rest of the steps of glycolysis and increases the ratio of ATP to ADP. The increase in ATP to ADP ratio causes an ATP-dependent or ATP-sensitive channel, a potassium channel, to close.

When that potassium channel closes, a depolarization occurs and that depolarization causes voltage-gated calcium channels to open and calcium moves into the cell. The increase in calcium in the beta cell causes secretory granules that have insulin already in them to be secreted, and then insulin is secreted outside the cell into the blood. Okay, so here is a better picture than the one that I just drew you, so we're going to kind of review here using this diagram.

So, glucose goes through the cell membrane into the cell via a glucose-2 transporter. Glucokinase then phosphorylates glucose into glucose 6-phospate, trapping the glucose into the cell. Glycolysis occurs and an increase in ATP to ADP ratio causes the ATP-sensitive potassium channel to close leading to a depolarization and calcium going into the cell via a voltage-gated calcium channel. Storage granules are then secreted and insulin is then released into the bloodstream. Remember that the metabolic actions of insulin are counteracted by glucagon and epinephrine. Remember that both of these receptors are coupled to stimulatory G-proteins. These proteins activate adenylate cyclase then increases cyclic-AMP and activates protein kinase A. Insulin receptors are not G-protein receptors, they are tyrosine kinase receptors, meaning that they have a phosphorylation activity on the cytoplasmic side of the protein and so insulin will bind to the alpha subunit of the protein and will propagate a signal through the cell.

I mentioned that in beta cells, glucose changes to glucose 6-phosphate via glucokinase. There's also another protein that does a similar action - hexokinase. So now I want to talk about the differences between hexokinase and glucokinase. Hexokinases are found more commonly in muscle and adipose tissue, whereas glucokinase is found more commonly in organs like the liver.

The differences between hexokinase and glucokinase are shown on the left side in this diagram. As you can see, glucokinase has a higher vmax and it also has a higher km. That means that at glucose concentrations that are higher, glucokinase has higher enzymatic activity than hexokinase. But as you can see in the graph, when glucose concentrations are low, hexokinase has an increase in enzymatic activity.

This is important because glucokinase is found in the liver, it has a higher vmax, and so at very high glucose concentrations, when glucose needs to be converted to glycogen in the liver, the enzyme is working very well. Whereas, at lower levels of glucose, when glucose needs to get into muscle and fat cells, hexokinase is working better. Also, to note, hexokinase is inhibited by glucose 6-phosphate so when there are really high levels of glucose 6-phosphate, hexokinase activity is diminished. But this is not the case with glucokinase, which is good because if there's a lot of glucose 6-phosphate but there are high amounts of blood glucose, we still need to get that glucose into the liver in order to make glycogen. So we just talked about hexokinase and glucokinase which are the first rate-limiting steps in glycolysis and how these two enzymes trap glucose in the cell by phosphorylation.

It also requires ATP that is converted into ADP. Glycolysis is really the backbone of glucose metabolism and there are branches out of glycolysis that kind of connect all of these pathways together. So, for example, we can make NADPH through the pentose phosphate pathway. We can make triacylglycerols, we can make mannose for signaling on the outside of the cell. There is also the uronic acid pathway, there's glycogen. Of course the citric acid cycle is connected to this as well.

Remember that glycolysis requires 2 ATP and for every 2 ATP that is used, 4 ATP is produced. Usually. There is aerobic glycolysis and there's anaerobic glycolysis. Aerobic glycolysis converts glucose into pyruvate, whereas in anaerobic glycolysis, glucose is converted to lactate. Both of these types of glycolysis occur in the cytosol of all body cells. Remember that anaerobic glycolysis is used when there is limited oxygen available because oxidative phosphorylation cannot occur. It is important to remember all of the different branches of glycolysis because when the body is in a starvation state, most of these pathways are diminished because the body is worried about making enough glucose.

So glycolysis is reversed and gluconeogenesis occurs. Now we will talk about the third step of glycolysis which is fructose 6-phosphate being converted into fructose 1,6-bisophosphate. This is done via phosphofructokinase-1. This is the rate-limiting step of glycolysis and as such, it's not only a regulated step, but it is the rate-limiting step. There are inhibitors and activators of this enzyme of PFK-1. Inhibitors include ATP and citrate which are indicators that there is enough energy where the cell does not need to make more ATP. Activators would be AMP and fructose 2,6-bisphoate.

These two molecules are indicators that ATP needs to be made and so glycolysis occurs. I like to think of glycolysis and gluconeogenesis as this teeter-totter. In one state, in a fasting state, gluconeogenesis would occur more often than glycolysis.

But it's not that gluconeogenesis is completely shut off. Even when glycolysis is up and running quite well, there is some part of the enzyme that is going the opposite direction for gluconeogenesis. I like to think of these rate-limiting steps as a waterfall, in that we have a high energy state that goes to a low energy state and it's very difficult to go back up. Meaning, that it is irreversible. It might be a little silly way to think about it, but it's kind of a way I like to think about these rate limiting steps - that they are irreversible because there's this waterfall and it's very difficult for the water or for the activity to go in the opposite direction.

So now we're going to talk about how fructose 2,6-bisphosphate controls the activity of PFK-1 as well as the activity of FBPase-1. FBPase-1 is fructose 1,6-bisphosphatase and it's the rate-limiting step of gluconeogenesis, which we'll talk about later. So it may be important or prudent for you to come back to this area of the lecture after we talk about gluconeogenesis, but I think it's important to talk about all of this once because it just makes things a little bit easier. So fructose 2,6-bisphosphate activates PFK-1. PFK-1 causes fructose 6-phosphate to be converted to fructose 1,6-bisphosphate, uses ATP and glycolysis is enhanced. Fructose 2,6-bisphosphate at high levels inhibits FBPase-1. FBPase-1 is inhibited so gluconeogenesis does not occur as often.

The teeter-totter is more towards glycolysis when fructose 2,6-bisphosphate levels are high. But, fructose 2,6-bisphosphate can be low at times. When fructose 2,6-bisphosphate levels are low, the opposite occurs so FBPase-1 is going to be activated more than PFK-1 and gluconeogenesis is going to occur. The concentration of fructose 2,6-bisphosphate is controlled by this enzyme here in the bottom right, which has PFK-2 and FBPase-2 activity. So remember, we just talked about PFK-1 and FBPase-1, now we're talking about PFK-2 and FBPase-2. I know it's a little confusing, but those are the names of the enzymes. So this enzyme is kind of like a two-for-one, two enzymes in one.

And insulin and glucagon control the activity of these two enzymes in this very large enzyme. So insulin generally dephosphorylates enzymes. Glucagon phosphorylates enzymes. So let's look here at this model. Insulin causes an increase in phosphoprotein phosphates activity. Phosphoprotein phosphate dephosphorylates this enzyme and activates the PFK-2 portion. When the PFK-2 portion is active, it stimulates an increase in fructose 2,6-bisphosphate activity, leading to glycolysis and inhibiting gluconeogenesis.

So insulin dephosphorylates. Glucagon phosphorylates indirectly, meaning that glucagon increases adenylate cyclase activity. Adenylate cyclase converts ATP into cyclic-AMP and cyclic AMP increases and cyclic-AMP dependent protein kinase phosphorylates the large enzyme, leading to PFK-2 being inactive and FBPase-2 being active. When this occurs, fructose 2,6-bisphosphate is decreased in concentration leading to an inhibition of glycolysis and a stimulation of gluconeogenesis. So, insulin dephosphorylates. Glucagon phosphorylates. So let's go over this one more time really quickly. Fructose 2,6-bisphosphate activates PFK-1 leading to glycolysis.

In addition, it inhibits FBPase-1 inhibiting gluconeogenesis at high concentrations. But at low concentrations, FBPase-1 is going to be more active than PFK-1, so it's going to swing the teeter-totter to gluconeogenesis. Remember this is all controlled by insulin and glucagon.

So at a feeding state meaning that there's a lot of glucose in the blood, insulin is going to lead to glycolysis. But in a starvation state, we need glucose in the blood we need glucose to get to those muscles and those fat cells. So glucagon is going to be in control. It's going to increase cyclic-AMP and it's going to enhance gluconeogenesis.

Alright, onto the ninth step of glycolysis which is activity of pyruvate kinase. Pyruvate kinase causes the conversion of phosphoenolpyruvate, easier said as PEP, to pyruvate. Okay, so inhibitors of this enzyme include ATP, alanine and PKA. Remember PKA is increased when cyclic-AMP activity is increased. So glucagon, this is glucagon we're talking about. Glucagon increases adenylate cyclase. Adenylate cyclase increases cyclic-AMP because ATP is converted to cyclic-AMP so cyclic-AMP leads to an increase in PKA activity.

So, glucagon, want glucose into the blood. We do not want glycolysis, we want gluconeogenesis. So, pyruvate kinase is going to be inhibited. Also, ATP and alanine indicate that the citric acid cycle is working, it's going on, so we don't need glycolysis anymore, we can convert glucose into something else.

Activators of pyruvate kinase include insulin. So remember that gluconeogenesis occurs in both the mitochondria and the cytosol and the liver is the primary source, the primary organ involved with gluconeogenesis but the kidneys and the epithelium of the small intestine have also been shown to have gluconeogenic activity and in order for glucose to get into the blood, we're going to need GLUT-2 transporters on all of these organs - on liver, kidneys, and small intestine epithelium. Okay, so this is a diagram of glycolysis. We just talked about the first, third, and ninth steps of glycolysis. The first step being glucokinase or hexokinase, the third step being PFK-1 and the ninth step being pyruvate kinase. So it's good to just look at this diagram of glycolysis when we're talking about gluconeogenesis, which is what we're going to talk about now. So there are three rate-controlling steps in glycolysis. So three kind of waterfalls, and we need to get up all three of them.

The first one we use two steps, and then the second and third waterfalls we use one step each. So, let's move down here and talk about the first step. The first step is pyruvate to oxaloacetate and this is done by pyruvate carboxylase. Remember that anytime you hear the word carboxylase, you think biotin. So, biotin is needed for this enzyme and this enzyme also requires ATP. Oxaloacetate is converted to phosphoenolpyruvate and this is done by phosphoenolpyruvatecarboxykinase. Pepck. And this converts oxaloacetate into phosphoenolpyruvate.

PEP-carboxykinase. The interesting thing about this enzyme is that it requires GTP. Always asked on the boards. So remember that - GTP. Let's go up here to the third step, we already talked about this third step earlier, the third step of glycolysis at least - the second waterfall that we need to go up. So we need to convert fructose 1,6-bisphosphate into fructose 6-phosphate and this is done by fructose 1,6-bisphosphatase. We already talked about how that is controlled by insulin and glucagon, right? The final step is conversion of glucose 6-phosphate to glucose.

This is done by glucose 6-phosphatase. And these are the four steps to overcome the three waterfalls. We have pyruvate carboxylase and we have PEP-carboxykinase that bypass step nine.

We have fructose 1,6-bisphosphase that bypasses step three, and remember this is the rate-limiting step. It dephosphorylates fructose 1,6-bisphosphate to produce fructose 6-phosphate. And finally, in order to bypass step one, we have glucose 6-phosphatase that dephosphorylates glucose 6-phosphate to produce glucose. Now remember that gluconeogenesis is how the body is able to maintain blood glucose levels in a fasting state with glucagon is king. And this occurs in the mitochondria and in the cytosol.

It requires 6 ATP. Okay, now on to glycogen metabolism. So we know that glycogen is a branched glucose polymer that's found in liver and skeletal muscle and it's the reserve supply of glucose. When we have a lot of glucose in the blood, we can convert that glucose into glycogen to store it. Remember that glucose 1-phosphate is the key metabolite that links glycogen synthesis to glycolysis. We know that glycogen synthase is the rate limiting enzyme of glycogen synthesis which is shown here on the diagram. And we're focusing on insulin, and glucagon, and epinephrine again. So insulin dephosphorylates and the dephosphorylated state of glycogen synthase is the active glycogen synthase.

So the active glycogen synthase is going to do exactly what it says, it's going to synthase or synthesize glycogen. Glucagon and epinephrine are going to phosphorylate glycogen synthase which is the inactive portion. So an inactive glycogen synthase is going to favor glycogenolysis, and that's done by glycogen phosphorylase, well it's done by a number of different enzymes, but we'll focus on glycogen phosphorylase because it's the rate-limiting enzyme and it's really how insulin and glucagon control it. So glucagon activates glycogen phosphorylase. It does the same thing as we've talked about in the past: glucagon leads to an increase in adenylate cyclase which leads to an increase in cyclic-AMP because that's the job of adneylate cyclase, to convert ATP into cyclic-AMP, and that causes an activation of protein kinase A. Protein kinase A is what phosphorylates glycogen phosphorylase. Remember that epinephrine also enhances glycogenolysis in both muscle and liver.

So again, glucagon is going to favor glycogenolysis via activation of glycogen phosphorylase whereas insulin is going to activate glycogen synthase, it's going to dephosphorylate it into its active state causing glycogen to be made. Okay, so we've now moved onto our last section in the video - lipids. So we'll first talk about fatty acid synthesis and it is controlled by the enzyme acetyl-CoA carboxylase. If you remember carboxylase, it's going to need biotin. And acetyl-CoA carboxylase converts acetyl-CoA (which has two carbons) into malonyl-CoA (which has three carbons). And as I said, it requires biotin, it requires CO2, and it requires ATP (which is converted to ADP). Stimulators of this enzyme include citrate and insulin, meaning we're at a fed state and we can convert acetyl-CoA, we don't need it, we can make fatty acids.

Inhibitors would include palmitate, AMP, glucagon, and epinephrine. All of these being that the body would be in a starvation state, that we need acetyl-CoA, we need it to make energy. And so all of these four molecules would inhibit the activity of acetyl-CoA carboxylase. We will now talk about lipolysis and when I talk about lipolysis I kind of want to talk about how epinephrine and growth hormone affect an adipose cell. So we're going to draw here an adipose cell and here we're going to draw a G-protein coupled receptor with Gs activity. So we're going to have epinephrine or growth hormone binding to this protein. This stimulates Gs, which then increases activity of adenylate cyclase. This converts ATP into cyclic AMP.

And what does cyclic AMP lead to? An increase in PKA. An PKA phosphorylates an enzyme, and this enzyme is the enzyme that helps lead to lipolysis. Lipolysis occurs in the fasting state when fat is required for energy. So hormone-sensitive lipase - PKA phosphorylates hormone-sensitive lipase. The phosphorylation of hormone-sensitive lipase causes triacylglycerides to be converted into fatty acids. And then fatty acids would go onto beta oxidation and making energy.

So that's the process. Epinephrine and growth hormone activate lipolysis by converting hormone-sensitive lipase into an active phosphorylated form by the activation of PKA. Insulin is going to activate a phosphatase, so that's going to take away the phosphate group because we know that insulin is a dephosphorylator. And that's going to inactivate (let's put insulin in here for us), activates the phosphatase which leads to inhibition of hormone-sensitive lipase. Just to mention lastly that glucagon has a weak effect on adipose tissue but it promotes gluconeogenesis in the liver which is supported by the energy from mobilized free fatty acids. So, glucagon does have a part in this but as far as working on adipose cells (this is an adipose cell, right?) epinephrine and growth hormone have more effect that glucagon does. Okay, so that just about finishes our video portion of diabetic ketoacidosis.

So questions to consider are what happens to gluconeogenesis, what happens to glycolysis, what happens to glycogen synthesis, what happens to lipolysis or fatty acid synthesis when we have glucose in the blood (these are red blood cells), when we have glucose in the blood that can't get out, meaning that either insulin does not exist or is very very low levels of insulin like in diabetes mellitus type 1. Or, we have these GLUT-4 receptors (right?) on adipose or in muscle and they're not working very well. They have become insensitive to insulin like in diabetes mellitus type 2. So, we have all of this glucose in the blood but we can't get it out. So in a sense, our body thinks that we're kind of in this starvation state, so what happens in a starvation state? What's going to happen to glucagon? What's going to happen to epinephrine? And what are the effects of this increase in glucose and what are the effects on the rest of the body? That's kind of what we're going to talk about in the lecture during class and you can read the lecture notes before class which will be very helpful for you to be able to answer the questions we'll talk about then. And so hopefully this was helpful for you, hopefully this kind of helps explain how glucagon and insulin affect fat metabolism and carbohydrate metabolism. We didn't talk too much about protein metabolism, but you can kind of get the idea that we're going to break down proteins into amino acids in a starvation state and proteins are going to be broken down, amino acids are going to be in the blood.

Whereas in a fed state, the opposite is going to occur. We didn't have enough time to talk about the protein metabolism, but I think you kind of get the picture we�ll focus mostly on fat and carbohydrate metabolism in the lecture anyway. So just kind of be remembering what protein and amino acids are going to be affected with this starvation state or pseudo-starvation state in diabetic ketoacidosis.


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