Voet D., Voet Ju.G. Biochemistry

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tion is initiated by the elimination of PP

from one farnesyl

pyrophosphate molecule to form an allylic carbocation at

C1 that is stabilized by a  interaction with an essential Tyr

residue (Fig. 25-53). The highly reactive electron-deficient

carbocation inserts into the electron-rich C2“C3 double

bond of the second molecule, yielding presqualene py-

rophosphate, a cyclopropylcarbinyl pyrophosphate.

Step II The rearrangement and reduction of presqua-

lene pyrophosphate by NADPH to form squalene.This re-

action involves the formation and rearrangement of a cy-

clopropylcarbinyl cation in a complex reaction sequence

called a 1–2–3 process (Fig. 25-54).

SQS, a monomeric protein, is anchored to the ER mem-

brane via a short C-terminal transmembrane domain, with

its active site facing the cytosol. This allows it to accept its

water-soluble substrates, farnesyl pyrophosphate and

NADPH, from the cytosol and release its hydrophobic

product, squalene, in the ER membrane.

Section 25-6. Cholesterol Metabolism 981

1

OPP

Presqualene pyrophosphate

1

OPP

1

OPP

Tertiary

carbocation

Allylic

carbocation

Farnesyl

pyrophosphate

Farnesyl

pyrophosphate

Figure 25-53 Proposed mechanism for the formation of

presqualene pyrophosphate from two farnesyl pyrophosphate

molecules by squalene synthase (Fig. 25-52, Step I). (1) The

pyrophosphate group on one farnesyl pyrophosphate leaves,

yielding an allylic carbocation.This reaction step is facilitated by

proton donation from the side chain of an essential Tyr residue,

which then stabilizes the allylic cation via –cation interactions.

(2) The C2“C3 double bond of the second farnesyl

pyrophosphate nucleophilically attacks the allylic carbocation

to form a tertiary carbocation at C3. (3) The abstraction of the

pro-S proton at C1¿ by the phenolate group of the essential Tyr

residue results in the formation of a C1¿¬C3 bond yielding

presqualene pyrophosphate.

1

OPP

Squalene

1

Presqualene pyrophosphate

Primary

cyclopropylcarbinyl

cation

1

H H

Tertiary

cyclopropylcarbinyl

cation

NADPH

NADP

Figure 25-54 Mechanism of rearrangement and reduction of

presqualene pyrophosphate to squalene as catalyzed by squalene

synthase (Fig. 25-52, Step II). (1) Presqualene’s pyrophosphate

group leaves, yielding a primary carbocation at C1. (2) The

electrons forming the C1¿¬C3 bond migrate to C1, forming

squalene’s C1¬C1¿ bond and a tertiary carbocation at C3. (3)

The process is completed by the addition of an NADPH-supplied

hydride ion to C1¿ and the formation of the C2“C3 double

bond.

JWCL281_c25_940-1018.qxd 6/8/10 8:59 AM Page 981

The X-ray structure of the 417-residue human SQS, in

complex with the inhibitor CP-320473,

determined by Jayvardhan Pandit, reveals that the protein

folds as a single domain with a large channel across one

face into which CP-320473 binds (Fig. 25-55). The channel

is lined with Asp and Arg residues that mutagenesis studies

indicate are involved in FPP binding. Of these, the con-

served Asp 80 and Asp 84 are implicated in binding Mg

2

ions that ligand an FPP pyrophosphate group. These Asp

residues are adjacent to Tyr 171, which forms the base of

the channel and which mutagenesis studies have identified

as the essential Tyr that is implicated in stabilizing the al-

lylic carbocation intermediate in Step I of the SQS reac-

tion. Step II of the SQS reaction requires that its highly re-

active carbocation intermediates be shielded from contact

with the aqueous solvent to prevent it from quenching the

reaction.This suggests that for Step II, the presqualene py-

rophosphate product of Step I moves deeper into the chan-

nel into a pocket that is lined with hydrophobic groups, in-

cluding Phe 288, whose mutation inactivates the enzyme.

This further suggests that Phe 288 functions to stabilize one

of the cationic intermediates in Step II (Fig. 25-54) through

–cation interactions.

d. Lanosterol Is Produced by Squalene Cyclization

Squalene, an open-chain C

hydrocarbon, is cyclized to

form the tetracyclic steroid skeleton in two steps. Squalene

CP-320473

COO



COO



CHNH

C(CH

)

epoxidase catalyzes the oxidation of squalene to form 2,3-

oxidosqualene (Fig. 25-56). Oxidosqualene cyclase (alter-

natively, lanosterol synthase) converts this epoxide to

lanosterol, the sterol precursor of cholesterol. The highly

exergonic reaction is a complex process involving cycliza-

tion of 2,3-oxidosqualene to a protosterol cation, via a

Class II mechanism involving protonation of the epoxide,

and rearrangement of this cation to lanosterol by a series

of 1,2 hydride and methyl shifts (Fig. 25-57). Note that this

reaction generates six of lanosterol’s seven chiral centers.

The X-ray structure of human oxidosqualene cyclase in

complex with lanosterol, determined by Armin Ruf, re-

veals that this 732-residue monomeric and monotopic [in-

tegral but not transmembrane] protein contains two struc-

turally similar domains named / barrels (Fig. 25-58a).

An / barrel consists of two concentric barrels of 6 helices

each, with the helices of the inner barrel largely parallel to

each other and antiparallel to those of the outer barrel

(much like an / barrel with the  strands of the inner

barrel replaced by helices but with only 6 / units rather

than 8 / units). The enzyme’s active site is located inside

an elongated central cavity (Fig. 25-58b) to which the

lanosterol is bound and which is accessible from the mem-

brane via a nonpolar channel through the enzyme’s

membrane-immersed part.

The interactions of 2,3-oxidosqualene with oxidosqua-

lene cyclase cause it to fold and react such that it only

forms lanosterol.The active site cavity is lined with several

conserved aromatic side chains that are suitably positioned

to stabilize the catalyzed reaction’s intermediate carboca-

tions, while shielding the cationic intermediates from pre-

mature quenching by either enzyme nucleophiles or water.

The cationic cascade (Fig. 25-57) is initiated by proton do-

nation by Asp 455 to the epoxide ring of the prefolded 2,3-

oxidosqualene, and is quenched by His 232 acting as a gen-

eral base.

The importance of the proper placement of residues for

the formation of the correct product was demonstrated by

site-directed mutagenesis: The conversion of Thr 384 in

982 Chapter 25. Lipid Metabolism

Figure 25-55 X-ray structure of human squalene synthase

(SQS) in complex with the inhibitor CP-320473. This inhibitor

together with the side chains of D80, D84, Y171, and F288 are

drawn in ball-and-stick form colored according to atom type

(inhibitor C green, protein C cyan, N blue, O red, and Cl magenta).

The protein is viewed looking into its central channel with the

putative active sites for Steps I and II of the catalyzed reaction

at the bottom and top of the cleft, respectively. The protein’s

C-terminal transmembrane segment (residues 371–417) together

with its N-terminal 30 residues were excised to facilitate its

crystallization, which did not affect its in vitro catalytic activity.

[Based on an X-ray structure by Jayvardhan Pandit, Pfizer

Central Research, Groton, Connecticut. PDBid 1EZF.]

JWCL281_c25_940-1018.qxd 6/8/10 8:59 AM Page 982

oxidosqualene cyclase to Tyr causes some misplacement of

the C8“C9 double bond, with 11% of the product having

a C9“C11 double bond and 10% having a 9-hydroxy

group; the double mutation, T384Y/V454I, increases the

yield of the C9“C11 double bond to 64%. This, together

with different ways of substrate folding, explains how en-

zymes homologous to oxidosqualene cyclase cause oxi-

dosqualene and squalene to form different products. In-

deed, the bacterial enzyme squalene–hopene synthase, a

homolog of oxidosqualene cyclase, synthesizes the C

compound hopene

from squalene. Its X-ray structure in complex with 2-

azasqualene (squalene with its C2 atom replaced N), deter-

mined by Georg Schulz, reveals that the enzyme, which

structurally resembles oxidosqualene cyclase, folds squa-

lene in the configuration that is predicted to yield hopene.

In this case, the cationic cyclase cascade is initiated by the

protonation of a double bond instead of an epoxide.

Hopene

Section 25-6. Cholesterol Metabolism 983

Squalene

+ O

2,3-Oxidosqualene

+ H

squalene

epoxidase

NADPH NADP

Enz

2,3-Oxidosqualene

Protosterol cation

Lanosterol

Figure 25-56 The squalene epoxidase reaction.

Figure 25-57 The oxidosqualene cyclase reaction. (1) 2,3-

Oxidosqualene is cyclized to the protosterol cation in a process

that is initiated by the enzyme-mediated protonation of the

squalene epoxide oxygen while this extended molecule is folded

in the manner predicted by Bloch and Woodward. The opening

of the epoxide leaves an electron-deficient center whose

migration drives the series of cyclizations that ultimately form

the protosterol cation. (2) A series of methyl and hydride

migrations yields a hypothesized intermediate carbocation at C8,

which then eliminates a proton from C9 to form the C8“C9

double bond of lanosterol.

JWCL281_c25_940-1018.qxd 4/20/10 1:59 PM Page 983

e. Cholesterol Is Synthesized from Lanosterol

Conversion of lanosterol to cholesterol (Fig. 25-59) is a

19-step process that we shall not explore in detail. It in-

volves an oxidation and loss of three methyl groups. The

first methyl group is removed as formate and the other two

are eliminated as CO

in reactions that all require NADPH

and O

. The enzymes involved in this process are embed-

ded in the ER membrane.

f. Cholesterol Is Transported in the Blood and Taken

Up by Cells in Lipoprotein Complexes

Transport and cellular uptake of cholesterol are de-

scribed in Section 12-5.To recapitulate, cholesterol synthe-

sized by the liver is either converted to bile salts for use in

the digestive process (Section 25-1) or esterified by acyl-

CoA:cholesterol acyltransferase (ACAT) to form choles-

teryl esters

which are secreted into the bloodstream as part of the

lipoprotein complexes called very low density lipoproteins

(VLDL). As the VLDL circulate, their component triacyl-

Cholesteryl ester

984 Chapter 25. Lipid Metabolism

Figure 25-58 X-ray structure of human oxidosqualene cyclase

in complex with its product lanosterol. The monomeric protein is

shown in ribbon form (a) viewed along the axis of its / barrels,

and (b) rotated 90° about the horizontal axis with respect to Part

a. The inner helices of its two / barrels are cyan, its outer

helices are purple, its nonpolar membrane-immersed portion is

orange, and the remainder of the protein is pink.The lanosterol,

which occupies the enzyme’s centrally located active site cavity, is

shown in Part a in space-filling form and in Part b in stick form

embedded in its semitransparent space-filling form, both with C

green and O red.The catalytically important side chains of His

232 and Asp 455, together with molecules of the detergent

-octylglucoside and fragments of lipids that coat the enzyme’s

membrane-immersed portion, are drawn in stick form with side

chain C magenta, lipid and detergent C yellow, and O red. The

arrow in Part b points to the membrane-immersed opening of

the enzyme’s active site cavity. [Based on an X-ray structure by

Armin Ruf, F. Hoffmann-La Roche AG, Basel, Switzerland.

PDBid 1W6K.]

(a)

(b)

JWCL281_c25_940-1018.qxd 6/8/10 9:00 AM Page 984

Section 25-6. Cholesterol Metabolism 985

Figure 25-59 The 19-reaction conversion of lanosterol to

cholesterol. [After Rilling, H.C. and Chayet, L.T., in Danielsson,

H. and Sjövall, J. (Eds.), Sterols and Bile Acids, p. 33, Elsevier

Lanosterol

NADPH

NAD(P)H

CHO

NAD(P)H

NADPH

CHO

NAD(P)H

OCH HOOC

NAD(P)H

NAD

HOH

NAD

NAD(P)H

NADPH

COOHCHO

NAD(P)H

NADPH

HCOOH

NADPH

Cholesterol

(1985), as modified by Bae, S.-H. and Paik,Y.-K., Biochem. J. 326,

609–616 (1997).]

JWCL281_c25_940-1018.qxd 4/20/10 1:59 PM Page 985

glycerols and most types of their apolipoproteins (Table

12-6) are removed in the capillaries of muscle and adipose

tissues, sequentially converting the VLDL to intermediate-

density lipoproteins (IDL) and then to low-density lipopro-

teins (LDL). Peripheral tissues normally obtain most

of their exogenous cholesterol from LDL by receptor-

mediated endocytosis (Fig. 25-60; Section 12-5Bc). Inside

the cell, cholesteryl esters are hydrolyzed by a lysosomal

lipase to free cholesterol, which is either incorporated into

cell membranes or reesterified by ACAT for storage as

cholesteryl ester droplets.

Dietary cholesterol, cholesteryl esters, and triacylglyc-

erols are transported in the blood by intestinally synthe-

sized lipoprotein complexes called chylomicrons. After re-

moval of their triacylglycerols at the peripheral tissues, the

resulting chylomicron remnants bind to specific liver cell

remnant receptors and are taken up by receptor-mediated

endocytosis in a manner similar to that of LDL.In the liver,

dietary cholesterol is either used in bile salt biosynthesis

(Section 25-6C) or packaged into VLDL for export. Liver

and peripheral tissues therefore have two ways of obtaining

cholesterol: They may either synthesize it from acetyl-CoA

986 Chapter 25. Lipid Metabolism

Endosome

Lysosome

Coated vesicle

Coated pit

LDL

particle

ApoB

protein

Golgi complex

LDL receptor

synthesis

Endoplasmic

reticulum

Nucleus

Cholesteryl

ester droplet

Amino acids

Recycling of

LDL receptors

LDL

receptors

Cholesteryl

ester

HMG-CoA reductase

synthesis

LDL receptor synthesis

ACAT

synthesis

Cholesterol

Figure 25-60 LDL receptor-mediated endocytosis in

mammalian cells. LDL receptor is synthesized on the

endoplasmic reticulum, processed in the Golgi complex, and

inserted into the plasma membrane as a component of coated

pits. LDL is specifically bound by the LDL receptor on the

coated pit and brought into the cell in endosomes that deliver

LDL to lysosomes while recycling LDL receptor to the plasma

membrane (Section 12-5Bc). Lysosomal degradation of LDL

releases cholesterol, whose presence decreases the rate of

synthesis of HMG-CoA reductase and LDL receptors (down

arrows) while increasing that of acyl-CoA:cholesterol

acyltransferase (ACAT; up arrow). [After Brown, M.S. and

Goldstein, J.L., Curr. Top. Cell. Reg. 26, 7 (1985).]

See the

Animated Figures

JWCL281_c25_940-1018.qxd 6/8/10 9:00 AM Page 986

by the de novo pathway we have just discussed, or they may

obtain it from the bloodstream by receptor-mediated endo-

cytosis. A small amount of cholesterol also enters cells by a

non-receptor-mediated pathway. Note however, that the

brain, which comprises ⬃2% of the human body mass but

contains ⬃30% of its cholesterol, must synthesize all of its

cholesterol because cholesterol cannot pass the blood-

brain barrier.

Cholesterol actually circulates back and forth between the

liver and peripheral tissues.While LDL transports cholesterol

from the liver, cholesterol is transported back to the liver by

high-density lipoproteins (HDL). Surplus cholesterol is dis-

posed of by the liver as bile salts, thereby protecting the body

from an overaccumulation of this water-insoluble substance.

B. Control of Cholesterol Biosynthesis

and Transport

Cholesterol biosynthesis and transport must be tightly reg-

ulated. There are three ways in which the cellular choles-

terol supply is maintained:

1. By regulating the activity of HMG-CoA reductase, the

enzyme catalyzing the rate-limiting step in the de novo choles-

terol biosynthesis pathway.This is accomplished in two ways:

(i) Short-term regulation of the enzyme’s catalytic ac-

tivity by (a) competitive inhibition, (b) allosteric ef-

fects, and (c) covalent modification involving re-

versible phosphorylation.

(ii) Long-term regulation of the enzyme’s concentra-

tion by modulating its rates of synthesis and

degradation.

2. By regulating the rate of LDL receptor synthesis, and

therefore the rate of cholesterol uptake. High intracellular

concentrations of cholesterol suppress LDL receptor syn-

thesis, whereas low cholesterol concentrations stimulate it.

3. By regulating the rate of esterification and hence the

removal of free cholesterol. ACAT, the enzyme that cat-

alyzes intracellular cholesterol esterification, is regulated

by reversible phosphorylation and by long-term control.

a. HMG-CoA Reductase Is the Primary Control

Site for Cholesterol Biosynthesis

HMG-CoA reductase is the rate-limiting enzyme in

cholesterol biosynthesis and, as therefore might be ex-

pected, constitutes the pathway’s main regulatory site. The

pathway branches after this reaction, however (Fig. 25-46);

ubiquinone, dolichol, farnesylated and geranylgeranylated

proteins, and isopentenyl adenosine are also essential,

albeit minor, products. HMG-CoA is therefore subject to

“multivalent” control, both long-term and short-term, in

order to coordinate the synthesis of all of these products.

b. Long-Term Feedback Regulation of HMG-CoA

Reductase Is Its Primary Means of Control

The main way in which HMG-CoA reductase is con-

trolled is by long-term feedback control of the amount of en-

zyme present in the cell. When either LDL–cholesterol or

mevalonate levels fall, the amount of HMG-CoA reduc-

tase present in the cell can rise as much as 200-fold, due to

an increase in enzyme synthesis combined with a decrease

in its degradation.When LDL–cholesterol or mevalonolac-

tone (an internal ester of mevalonate that is hydrolyzed to

mevalonate and metabolized in the cell)

are added back to a cell, these effects are reversed.

The mechanism by which cholesterol serves to control

the expression of the ⬎20 genes involved in its biosyn-

thesis and uptake, such as those encoding HMG-CoA re-

ductase and the LDL receptor, has been elucidated by

Michael Brown and Joseph Goldstein. These genes all

contain a DNA sequence upstream from the transcrip-

tion initiation site called the sterol regulatory element

(SRE). In order for these genes to be transcribed, a spe-

cific transcription factor, the sterol regulatory element

binding protein (SREBP), must bind to the SRE (eu-

karyotic gene expression is discussed in Section 34-3).

SREBP is synthesized as an integral membrane protein

that, when the cholesterol concentration is sufficiently

high (Fig. 25-61a), resides in the ER membrane in com-

plex with SREBP cleavage-activating protein (SCAP)

and a protein named Insig. SREBP (⬃1160 residues)

consists of three domains (Fig. 25-61a): (1) an ⬃480-

residue cytosolic N-terminal domain that is a member of

the basic helix–loop–helix/leucine zipper (bHLH/Z)

family of transcription factors (Section 34-3Br), which

specifically binds to SREs; (2) an ⬃90-residue transmem-

brane (TM) domain consisting of two TM helices con-

nected by an ⬃30-residue hydrophilic luminal loop; and

(3) an ⬃590-residue cytosolic C-terminal regulatory do-

main. SCAP (1276 residues) consists of two domains: (1)

a 730-residue N-terminal domain that contains eight TM

helices; and (2) a 546-residue cytosolic C-terminal do-

main that contains five copies of the protein–protein in-

teraction motif known as a WD repeat (also called a

WD40 sequence motif because it is ⬃40 residues long;

Section 19-2Cc) and which presumably forms a 5-bladed

␤ propeller similar to the 7-bladed ␤ propeller of the

␤

subunit (Fig. 19-19b). Insig (named to denote insulin-

induced gene, although the insulin effect was later found

to be indirect) consists mainly of six TM helices. SCAP

and SREBP associate through the interaction of the

SCAP’s regulatory domain with SREBP’s WD domain

(Fig. 25-61a).

SCAP functions as a sterol sensor. An ⬃170-residue

segment of its TM domain, the sterol-sensing domain, in-

teracts with sterols although how it does so is unknown.

When the cholesterol in the ER membrane is depleted

(Fig. 25-61b), SCAP changes conformation and then

Mevalonolactone

Section 25-6. Cholesterol Metabolism 987

JWCL281_c25_940-1018.qxd 6/18/10 7:51 AM Page 987

releases Insig, which allows SCAP to bind to the Sec24

cargo-binding subunit of the COPII coated vesicle protein

(Section 12-4Ca).The COPII vesicle then escorts its bound

SREBP–SCAP complex to the Golgi apparatus.

In the Golgi apparatus, SREBP is sequentially cleaved

by two membrane-bound proteases (Fig. 25-61b). Site-1

protease (S1P), a serine protease of the subtilisin family,

cleaves SREBP in the luminal loop that connects its two

transmembrane helices but only when it is associated

with SCAP.This cleavage exposes a peptide bond located

3 residues from the beginning of SREBP’s N-terminal

TM helix to cleavage by site-2 protease (S2P), a zinc met-

alloprotease. This releases the bHLH/Z domain to mi-

grate to the nucleus where it activates the transcription

of its target genes. The cholesterol level in the cell

thereby rises until SCAP no longer induces the transloca-

tion of SREBP to the Golgi, a classic case of feedback in-

hibition.

This complex regulatory pathway was elucidated, in

part, through the generation of several lines of transgenic

mice that overexpress one or another of the foregoing pro-

teins, and knockout mice that lack one or another of these

988 Chapter 25. Lipid Metabolism

SREBPSCAP

Cytosol

COPII

Insig

Transport

to Golgi

Transport

to nucleus

Golgi

apparatus

Lumen

Nucleus

SRE

S1P

S2P

Serine protease Metalloprotease

bHLH

bHLHReg.

SREBPSCAP

Cytosol

Insig

Lumen

COPII

High cholesterol

Low cholesterol

WD bHLHReg.

WD bHLHReg. bHLH

(a)

(b)

Figure 25-61 The cholesterol-mediated proteolytic activation

of SREBP. (a) When cholesterol levels in the cell are high, the

Insig–SCAP–SREBP complex resides in the ER membrane.

(b) When cholesterol levels are low, SCAP releases Insig and binds

COPII protein.The SCAP–SREBP complex is then transported,

via COPII-coated vesicles, to the Golgi apparatus, where SREBP

undergoes sequential proteolytic cleavage by the

membrane-bound proteases S1P and S2P. This releases SREBP’s

bHLH/Z-containing N-terminal domain, which enters the

nucleus where it binds to the SREs of its target genes, thereby

inducing their transcription. [After Goldstein, J., Rawson, R.B.,

and Brown, M., Arch. Biochem. Biophys. 397, 139 (2002).]

JWCL281_c25_940-1018.qxd 8/9/10 10:21 AM Page 988

proteins. For example, knockout mice lacking either SCAP

or S1P in their livers have decreased expression of both

HMG-CoA reductase and LDL receptor, even when fed a

cholesterol-deficient diet. In contrast, transgenic mice that

overexpress SREBP or SCAP have greatly increased ex-

pression of the foregoing proteins. In fact, animals overpro-

ducing only the bHLH/Z domain of SREBP have mas-

sively enlarged livers (up to 4-fold larger than normal) due

to engorgement with triacylglycerols and cholesteryl esters

and yet they continue to transcribe SREBP’s target genes

such that their mRNA levels are up to 75-fold greater than

normal. Many individuals suffering from obesity or dia-

betes caused by insulin resistance (type 2 diabetes; Section

27-4B) have fatty livers, which in some cases leads to liver

failure. Fatty livers due to insulin resistance appear to be

caused by elevated levels of SREBP in response to ele-

vated insulin levels.

The level of HMG-CoA reductase also responds to

the level of the cholesterol precursor lanosterol (Section

25-6Ad). HMG-CoA reductase’s ER membrane-bound

N-terminal domain contains eight TM helices, whereas its

C-terminal domain, which contains its active site and is

linked to the N-terminal domain via a flexible Pro-rich se-

quence, projects into the cytosol. Insig binds to an enzy-

matic complex that marks proteins for degradation by co-

valently linking them to the protein ubiquitin (Section

32-6B). When lanosterol accumulates in the ER mem-

brane, the N-terminal domain of HMG-CoA reductase

also binds to Insig,and is thus marked for destruction. Con-

sequently, the 12-hour half-life of HMG-CoA reductase

in sterol-deprived cells decreases to 1 hour when sterols

are plentiful.

c. Regulation of HMG-CoA Reductase by Covalent

Modification Is a Means of Cellular Energy

Conservation

HMG-CoA reductase exists in interconvertible more ac-

tive and less active forms, as do glycogen phosphorylase

(Section 18-3Ca), glycogen synthase (Section 18-3D), pyru-

vate dehydrogenase (Section 21-2Cb), and acetyl-CoA car-

boxylase (Section 25-4Ba), among others. The unmodified

form of HMG-CoA reductase is more active and the phos-

phorylated form is less active. HMG-CoA reductase is

phosphorylated (inactivated) at its Ser 871 in a bicyclic cas-

cade system by the covalently modifiable enzyme AMP-

dependent protein kinase (AMPK), which, as we saw in

Section 25-4Ba, also acts on acetyl-CoA carboxylase [in

this context, this enzyme was originally named HMG-CoA

reductase kinase (RK), until it was found to be identical to

AMPK]. It appears that this control is exerted to conserve

energy when ATP levels fall and AMP levels rise, by in-

hibiting biosynthetic pathways. This hypothesis was tested

by Brown and Goldstein, who used genetic engineering

techniques to produce hamster cells containing a mutant

HMG-CoA reductase with Ala replacing Ser 871 and

therefore incapable of phosphorylation control.These cells

respond normally to feedback regulation of cholesterol

biosynthesis by LDL–cholesterol and mevalonate but, un-

Section 25-6. Cholesterol Metabolism 989

like normal cells, do not decrease their synthesis of choles-

terol on ATP depletion, supporting the idea that control of

HMG-CoA reductase by phosphorylation is involved in

energy conservation.

d. LDL Receptor Activity Controls

Cholesterol Homeostasis

LDL receptors clearly play an important role in the

maintenance of plasma LDL–cholesterol levels. In normal

individuals, about half of the IDL formed from the VLDL

reenters the liver through LDL receptor-mediated endocy-

tosis (IDL and LDL both contain apolipoproteins that

specifically bind to the LDL receptor; Section 12-5Bc).The

remaining IDL are converted to LDL (Fig. 25-62a). The

serum concentration of LDL therefore depends on the rate

at which liver removes IDL from the circulation, which, in

turn, depends on the number of functioning LDL receptors

on the liver cell surface.

High blood cholesterol (hypercholesterolemia), which

results from the overproduction and/or underutilization of

LDL, is known to be caused by either of two metabolic

irregularities: (1) the genetic disease familial hyper-

cholesterolemia (FH) or (2) the consumption of a high-

cholesterol diet. FH is a dominant genetic defect that

results in a deficiency of functional LDL receptors (Section

12-5Ca). Homozygotes for this disorder lack functional

LDL receptors, so their cells can absorb neither IDL nor

LDL by receptor-mediated endocytosis.The increased con-

centration of IDL in the bloodstream leads to a correspon-

ding increase in LDL, which is, of course, underutilized

since it cannot be taken up by the cells (Fig. 25-62b). FH

homozygotes therefore have plasma LDL–cholesterol lev-

els three to five times higher than average. FH heterozy-

gotes, which are far more common, have about half of the

normal number of functional LDL receptors and plasma

LDL–cholesterol levels about twice the average.

The long-term ingestion of a high-cholesterol diet has an ef-

fect similar, although not as extreme, as FH (Fig. 25-62c). Ex-

cessive dietary cholesterol enters the liver cells in chylomi-

cron remnants and represses the synthesis of LDL receptor

protein. The resulting insufficiency of LDL receptors on the

liver cell surface has consequences similar to those of FH.

LDL receptor deficiency, whether of genetic or dietary

origin, raises the LDL level by two mechanisms: (1) in-

creased LDL production resulting from decreased IDL

uptake and (2) decreased LDL uptake. Two strategies for

reversing these conditions (besides maintaining a low-

cholesterol diet) are being used in humans:

1. Ingestion of anion exchange resins (Section 6-3A) that

bind bile salts, thereby preventing their intestinal absorption

(resins are insoluble in water). Bile salts, which are derived

from cholesterol, are normally efficiently recycled by the

liver (Section 25-6C). Elimination of resin-bound bile salts in

the feces forces the liver to convert more cholesterol to bile

salts than otherwise. The consequent decrease in the serum

cholesterol concentration induces synthesis of LDL recep-

tors (of course, not in FH homozygotes). Unfortunately, the

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decreased serum cholesterol level also induces the synthesis

of HMG-CoA reductase, which increases the rate of choles-

terol biosynthesis. Ingestion of bile salt–binding resins such

as cholestyramine (sold as Questran) therefore provides only

a 15 to 20% drop in serum cholesterol levels.

2. Treatment with competitive inhibitors of HMG-CoA

reductase. These include (Fig. 25-63) the fungal derivatives

lovastatin (also called mevinolin and sold as Mevacor),

pravastatin (Pravachol), and simvastatin (Zocor) as well as

the synthetic inhibitor atorvastatin (Lipitor), compounds

990 Chapter 25. Lipid Metabolism

Figure 25-62 Control of plasma LDL production and uptake

by liver LDL receptors. (a) In normal human subjects, VLDL is

secreted by the liver and converted to IDL in the capillaries of

the peripheral tissues.About half of the plasma IDL particles

bind to the LDL receptor and are taken up by the liver.The

remainder are converted to LDL at the peripheral tissues. (b) In

individuals with familial hypercholesterolemia (FH), liver LDL

receptors are diminished or eliminated because of a genetic

Liver

VLDL

LDL

IDL

VLDL

Dietary

cholesterol

LDL

IDL

Capillary

lipoprotein

lipase

Free fatty

acids

(a) Normal

Normal LDL

receptor

function

Cholesterol repression

of LDL receptor

synthesis

VLDL

LDL

IDL

(b) Familial hypercholesterolemia

Genetically defective

LDL receptor

synthesis

lipoprotein

lipase

Free fatty

acids

lipoprotein

lipase

Free fatty

acids

defect. (c) In normal individuals who have a long-term

high-cholesterol diet, the liver is filled with cholesterol, which

represses the rate of LDL receptor production. Receptor

deficiency, whether of genetic or dietary cause, raises the plasma

LDL level by increasing the rate of LDL production and

decreasing the rate of LDL uptake. [After Goldstein, J.L. and

Brown, M.S., J. Lipid Res. 25, 1457 (1984).]

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