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  • JOANNE STUBBE: Where we were at at the end of the last lecture

  • was trying to figure out what do we

  • do with the fact that cholesterol--

  • its solubility is five micromolar.

  • Yet if you look inside your blood,

  • the levels would be 5 millimolar.

  • And so the question is, how does it gets transported?

  • And it gets transported in a complex fashion.

  • We need to deal with that with any kind of very

  • insoluble lipophilic materials.

  • And I briefly introduced you to lipoproteins,

  • which are mixtures of different kinds of lipids,

  • triacylglycerols, phospholipids, cholesterol,

  • cholesterol esters.

  • And the key question we learned in the first couple

  • lectures that cholesterol could be biosynthesized.

  • And what we started focusing on in the last lecture

  • was that it can be taken up by the diet.

  • That's what we're focusing on now.

  • And then after we do a little more background,

  • then how is it taken up and then how is this all regulated?

  • How do you control biosynthesis versus cholesterol

  • from the diet.

  • What are the sort of major mechanisms?

  • So at the end of the last lecture

  • I'd given you a second picture.

  • And the PowerPoint-- the original PowerPoint

  • didn't have this figure.

  • This is taken out of a new Voet and Voet--

  • the newest Voet and Voet-- which I think better

  • describes what's going on.

  • But really sort of what you need to know

  • is you form these particles, chylomicrons,

  • if you look at the handout I gave

  • you have lots of proteins, all kinds of lipids, cholesterol.

  • And they get into the bloodstream

  • and they pass off as they go through adipocytes

  • or as they go through muscle.

  • The surface of these cells have lipases, phospholipases

  • that can clip off the fatty acids

  • that you need for metabolism at most cells.

  • And what happens is the size of these particles just change.

  • And so in the end, you remove the triacylglycerols

  • and you remove phospholipids.

  • And what you're left with is more of a cholesterol.

  • And that-- and so what happens is the chylomicrons change

  • size.

  • They call them the remnants.

  • And there are receptors on liver cells, which

  • can take up these remnants, these lipoprotein remnants.

  • And then they repackage them into other lipoproteins.

  • And again, the differences in the lipoproteins

  • we talked about very briefly, we have an outline.

  • Somebody measured these with a--

  • again, they're variable, but they're based on density.

  • And so the liver repackages these things

  • to a particle that's very low density, lipoprotein.

  • And then again, they can dump off components

  • into the tissues where you can use the lipids to do

  • metabolism, changing the size, intermediate density,

  • eventually low density lipoprotein which

  • is what we're focused on now.

  • And then today what we're focused

  • on is how does the low density lipoprotein get taken up

  • by the liver?

  • And also, can it get taken up by other kinds of cells?

  • And if you have excess cholesterol produced

  • in any of these extrahepatic cells,

  • it can be taken up to form particles called high density

  • lipoproteins.

  • And they can come back.

  • So they act as cholesterol scavengers,

  • come back and deliver it back into the liver

  • by a mechanism that is really different from what we're going

  • to be talking about today.

  • So that's the overview picture.

  • And so what I want to do now is focus

  • on the question, why do we care about cholesterol

  • and what was the motivator for Brown

  • and Goldstein's discovery of the low density lipoprotein

  • receptor.

  • So this is the motivator.

  • They were seeing when they were at medical school,

  • a number of children that presented at an early age.

  • These guys were six and eight.

  • And the way they present, if they turn out

  • to have both genes, both copies of the gene

  • are messed up for low density lipoprotein

  • receptor, that's called familial hypercholesterolemia.

  • The way they present is they have these little xanthomases

  • that are apparently yellow.

  • And what they are is they're full of cholesterol.

  • OK, and so if you have someone that's heterozygous

  • rather than homozygous--

  • these guys are homozygous--

  • you still see these but you see it

  • at a much later time in their life.

  • And so again, what it is, it's a function of the fact

  • that you have too much cholesterol

  • and this is the way--

  • one of the ways-- it manifests itself.

  • The second way it manifests itself

  • is if you look at the concentration of low density

  • lipoprotein and the plasma, which

  • is given in milligrams for 100 mils, what you see

  • is the concentrations of cholesterol

  • are actually 5 to 10 times higher.

  • So that's the manifestation.

  • And children that manifest at this early age

  • die of heart attacks by the time they're 30.

  • And so this was the motivator.

  • They were trying to figure out what is the basis

  • or bases for this disease.

  • So that's what I said.

  • This is a dominant effect.

  • At the time, the gene or genes responsible for this

  • were not known.

  • It turns out from the data that I've gotten from some paper,

  • one in 500 people are heterozygotes.

  • That's quite prevalent, actually.

  • But the ones that manifest themselves

  • in this really terrible way early on

  • is something like one in a million.

  • And so-- but even the heterozygotes,

  • Brown and Goldstein study all of these people,

  • also manifest in this way.

  • They have elevated cholesterol levels.

  • And so this was is a huge problem.

  • And so they decided they wanted to really devote

  • their life to it.

  • And I think they didn't know this in the beginning,

  • but it's really associated with one gene.

  • Most diseases are much more complicated than that.

  • And so I think because of the, quote, "simplicity" unquote,

  • you'll see it's not so simple, they

  • were able to make progress.

  • And these experiments were carried out really

  • sort of in the--

  • started in the 1970s.

  • So I think Brown and Goldstein-- we talked about the cholesterol

  • biosynthetic pathway.

  • And we talked about what was rate limiting.

  • So hopefully you all know that the rate limiting step

  • is the reduction of hydroxymethylglutaryl CoA down.

  • So the CoA is reduced all the way down to an alcohol

  • and that product is mevalonic acid.

  • And if you can't remember this, you

  • should pull out the biosynthetic pathway.

  • And that was proposed to be by other people working

  • in this field to be the rate limiting

  • step in this overall process.

  • And when you take an introductory course

  • in biochemistry, you talk about regulation.

  • I guess it depends on who's teaching it,

  • how much you talk about regulation.

  • But of course, one of the major mechanisms of regulation

  • that's sort of easy to understand in some fashion,

  • is that oftentimes the end product of a pathway

  • can come back way at the beginning

  • and inhibit the pathway.

  • So that's called feedback inhibition.

  • We saw that cholesterol biosynthesis was 30 steps.

  • And if you go back and you look at the pathway, you know,

  • I think this is step four or five.

  • I can't remember which one it is.

  • And so the model was--

  • and there was some evidence that suggested that

  • from what had been done in the literature--

  • that cholesterol was potentially acting as a feedback inhibitor.

  • And that's what their original working hypothesis was.

  • So the hypothesis was--

  • this is how they started it out.

  • And what we'll do is just look at a few experiments

  • of how they were trying to test their hypothesis

  • and then how they change their hypothesis to come up

  • with a new model for cholesterol regulation.

  • So you start out with acetyl CoA and you

  • go through mevalonic acid.

  • And then we get to cholesterol.

  • And so the model was that--

  • this is HMG reductase-- that this was a feedback inhibitor.

  • And that it inhibited by allosteric regulation.

  • And that's true of many pathways.

  • And often, that's one out of many mechanisms

  • that are involved in regulation.

  • So the first problem they faced--

  • and for those of you who want to read

  • about this in more detail, the original experiments,

  • I'm just going to present a few simple experiments

  • and I'm going to present them in a simple way.

  • OK, everything with human cells is more complicated

  • than the way I'm presenting it.

  • But for those of you would like to read a little bit more

  • about the actual experiments, there

  • are two papers that I think are particularly compelling.

  • And in previous years, I've actually used these papers

  • in recitation.

  • OK, so this is one of them.

  • I'll put the other one up later on so

  • that you can look at the detail, more

  • about the experimental details.

  • And I think in these particular experiments, what you're

  • being introduced to, which most students don't experience,

  • is the fact that you have--

  • all you do with these insoluble membrane-like proteins

  • and how do you deal with membrane proteins.

  • Most of us-- I haven't had any experience with this at all.

  • So this week's recitation, for example,

  • sort of shows you what they had to go through

  • to be able to answer these questions.

  • And it's complicated.

  • And I think reading the experimental details

  • in the end, if you're going to do something like this,

  • this provides a nice blueprint of how you try--

  • how you try to design experiments.

  • And you'll see some of the complexity

  • from the few experiments I'm just going to briefly describe.

  • OK, so what they needed was a model system.

  • And of course, you can't do experiments on humans.

  • So what they wanted to do was have some kind

  • of tissue culture system.

  • So they wanted a model system.

  • And there was some evidence in the literature

  • that human fibroblast skin cells were actually

  • able to biosynthesize cholesterol.

  • So they wanted to ask the question,

  • do these skin cells recapitulate what

  • people had seen from the biological studies in humans?

  • And so the first experiments I'll show you,

  • does recapitulate that.

  • It didn't have to.

  • But then this became their model,

  • human fibroblast cells became the model

  • for which they're carrying out all of these experiments

  • that we're going to very briefly look at.

  • OK, so the experiments, I think, are simple,

  • at least on the surface.

  • Although I think it wasn't so easy to figure out

  • how to do these experiments.

  • So what they wanted to do, they had patients--

  • whoops.

  • I didn't want to do that.

  • Anyhow, sorry I'm wasting time.

  • OK, this patient is JD.

  • And all of the experiments I'm going to show you is JD.

  • But they had 25 other patients.

  • And what you'll see is they all manifest themselves

  • in different ways.

  • And we're going to see that that, in the end,