[SOUND] Welcome, as we continue to think about the different components of milk and how they're synthesized and secreted from the mammary epithelial cells, we're going to focus, this particular video on fat. And here it reminds you several things. First of all 98% of the milk fat, the fat found in milk is tricylglyceride or tricylglyceride. So, to remind you the structure of tricylglyceride, we have glycerol which is three carbon chain here basically. And those are esterified to fatty acids, and I've just indicated that 1, 2 and 3, fa for fatty acid. They could be the same fatty acids or different fatty acids and so on. So that's not as relevant as at the moment. The key is, tri meaning three of them, acylglycerol so it's glycerol this part here and then three fatty acids. So what we need to do is, we think about fat synthesis and secretion is. We need to think about how do we synthesize fatty acids, first of all. And secondly, how do we synthesize this triacylglyceride. So we're going to start out with how are fatty acids synthesized in the mammary epithelial cells. So to remind you what the mammary epithelial cells look like, this is one here, electron micrograph. Again, we have the nucleus here. The cell membrane would kind of come down like this. So the basal side of the cell would be down here. Apical slide up here. So this is a lumen up here. We have a nice milk fat droplet here. We're going to talk a little bit later about how this milk fat droplet is formed and then released out into the milk. The secretory vesicles Casein Micelles are these really darkest tiny dots. Again we don't necessarily see very well in this particular image the Golgi apparatus and so on. So this is just to remind you kind of setting up how the cell is organized. Let's go to the next slide. And this is one we've presented in some of the other slides for lactone synthesis, protein synthesis We've talked about secretory vesicles, golgi apparatus, rough endoplasmic reticulum, and we're going to get rid of that stuff because it really doesn't have a lot to do directly with milk fat synthesis. So let's go to the next slide. What we do need to do, though, is think about cytosol. So out in the cytosol are a number of factors. One of those factors is called fatty acid synthetase. We're looking at fatty acid synthesis now, so breaking down, again we'll talk about triglycerides synthesis in another part of the video. I've drawn it very specifically, or put this symbol here very specifically because, what's really interesting about this fatty acid synthetase molecule is it's a very large molecule, one single elastic chain, very large one, but it has about seven enzyme activities to it. So a whole bunch of enzyme activities integrated into this protein and sometimes people call this a daisy wheel. We are getting to kind of why it is called a daisy wheel in a moment here. So fatty acid synthesis is a very large enzyme molecule or multi-enzymatic molecule out in the cytosol. So that's where things are happening. Precursors are taken up by the cell. Let's go ahead and go to the next cell, the next slide. One of those would be acetate or some other precursors. Those are some of those conversion to Acetyl CoA. And that's then converted to malenyl CoA. So the CoA part is really just high energy. So Acetate by itself, malenite by itself are not going to do very much. But if you put on these CoA collating high energy moieties, it allows them to go through the reactions and do the things that they are supposed to do. Let's go to the next slide. So what we have is an enzyme called Acetyl-CoA Carboxylase. And that's the enzyme that converts Acetyl-CoA to this high energy Malonyl-CoA. And it is the rate limiting step for fatty acid synthesis. Not just in the mammary gland but in other tissues as well. So it is a rate-limiting step. So this is a very, very important enzyme in terms of fat synthesis in again, pretty much any cell. And certain in the mammary epithelial cell. And again it's this reaction right here. This arrow, the enzyme is causing conversion of Acetyle-CoA to Malonyl-CoA is Acetyl-CoA Carboxylase. We find then is that these two components then combine with the fatty acid synthetase molecule, puts them together and so just point out a couple of things. This sis a two carbon chain. This is a three carbon, two plus three is five. Most fatty acids, not all, but many fatty acids are even numbers. So we start out with five, what we're going to find, let's go to the next slide, is that there's a series of reactions. This five carbon first product from this is going to go around and be changed. Different kinds of reactions as it goes around, this fatty acid synthetase molecule. A carbon dioxide comes off. So you're left with four carbons. So as it keep comes around to this point. Let's go to the next slide. It's four carbons. Add three, that makes seven. Carbon dioxide comes off as this goes around to reaction again. You get six. It keeps going around and around and around. So that's why fatty acids, most of the fatty acids are even numbers. It's this two plus three is five, goes around kicks off a carbon dioxide. You get an even number, add three here. Goes around, adds three, and so on and so forth. And each time it goes around, a carbon dioxide goes off and you end up with an even number fatty acid. At whatever point it's going to come off, and it depends upon the species. It depends upon the circumstances as to whether it comes off as a short chain fatty acid, say it's 6, or a medium chain, at say, 12 carbons, or a longer chain at 16 or 18 carbons. There's other, again, other enzymes in the cytosol that help to cleave that thing off. It's associated with something called ACP here or Acyl-carrier protein. Most cells don't like fatty acids by themselves. They like to have them bound or something like that. Sometimes they're actually toxic to cells. So very often you'll find fatty acids in a cell associated with some sort of a protein. This particular case is called Acyl-carrier protein. So it's just fatty acid associated with a protein that curates it around through the cell. So there's the Fa-ACP. So now we've produced our fatty acids. Again, typically they're even numbered carbons. And, depending upon the species. Again, cows have short and medium chain and long chain fatty acids in their milk fat. Pigs, for example, don't have very much short and medium chain at all. They have really more longer chain fatty acids. So, it depends upon the species as to how many times a thing would go around, in terms of when it is kicked off of fatty acids of the face molecule. Are releasing the fatty acid ACP. We just finished part of the video looking at fatty acid synthesis. And that is the fatty acids that are synthesized inside the mammary epithelial cells. We call that De novo synthesis, when it's actually produced in the cell. From those precursors, the acetate, and other kinds of things like that. Now we want to do is we want to look at synthesis of the triglyceride. So let's take a quick look at that. So triacylglyceride synthesis is what we're going to talk about now. Recall that the fatty acid, ACP is out in cytosol, and the blood we have triglycerides usually in the form of lipoproteins. So very low density lipoproteins,kiomicrons, other kinds of things. Depends on where those are coming from. From other parts of the body they're flowing through here. And you have an enzyme in the blood. And on the inside of the capillary is called lipoprotein lipase. That starts to hydrolyse that triglyceride that's associated with those lipoproteins. So you end up with three fatty acids, monoacylglycerides, which are the glycerol, the glycerol with one fatty acid. Diacylglycerides glycerol with two fatty acids on it. And then of course, the free glyceride itself, glycerol. Those are taken up into the cell. Let's go to the next slide. So, what we have is a whole bunch of precursors here that can be used to produce triglycerides. So essentially what's happened here is that the fat that's in the blood is hydrolyzed, broken down into it's components. It's components are taken it to the cell and then combined with the fatty acids that are produced inside the cell. We're going to put all that back together again and then that's secreted as the milk fat. So let's go to the next slide. That process of making the triglyceride in the cell occurs on the smooth endoplasmic reticulum. We talked about rough endoplasmic reticulum with the ribosomes in terms of protein synthesis, smooth endoplasmic reticulum. It's called smooth, because there are no ribosomes associated with it, and I've just drawn three lines here to demonstrate that. And it's actually being produced on the cytosolic side of the membrane of the smooth endoplasmic reticulum. That's where the enzymes are that are making between visceral and the fatty acid. So let's go to the next slide. As those fatty acids, excuse me, those triglycerides are being produced on the cytosolic side of this membrane. Of course, they're hydrophobic, and so they don't like water. And so they're going to kind of glom together. And as I've kind of indicated that here by these yellow blobs as those are then released into the cytosol, go to the next slide. Again they are hydrophobic so they tend to kind of coalesce together over time, next slide. And then gradually they work their way up to the atypical membrane, they get larger and larger and larger as more and more of these micro believes basically of a fat are coming together and forming these milk fat globules. So inside the cell, they're called milk fat globules. Outside the cell, they're called milk fat droplets. Now I've kind of indicated here that they're just floating up in the cells. They're actually surrounded by several proteins. One particular protein is called butyrophilin. And so there is a protein coating. Not a membrane as such, but a protein coating around these fat globules. As they come to this apical surface, what they'll do is they'll kind of push their way through. And are surrounded by the membrane. Go to the next slide. And you can just barely see here, I probably should have highlighted it more, a white circle around this so they kind of push their way through. The way I usually explain this to students, if you're standing inside a balloon and you push your fist through the balloon, the balloon wraps around your fist. Then, eventually, in this case, it'll pinch off, the milk fat droplet will then be in the lumen. The milk fat droplet has this core of trisoglycerol. This right, surrounded by that butyrophilin. But then that's surrounded by the apical membrane, or something very similar to the apical membrane, as it pushes through there. So this is our trisoglyceride synthesis, how it synthesizes smooth endoplasmic reticulum and then the fat globules inside the cell. And then once they're released and they're membrane-bound, those are the fat droplets.
