1. health and fitness
2. disease
3. cholesterol

REFLECTIONS
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 287, NO. 27, pp. 22418 –22435, June 29, 2012
© 2012 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.
Scientific Side Trips:
Six Excursions from the
Beaten Path
Published, JBC Papers in Press, May 14, 2012, DOI 10.1074/jbc.X112.381681
Michael S. Brown and Joseph L. Goldstein
Origin of a 40-Year Research Partnership
ur 40-year research partnership began on a sunny June day in 1966 at the Massachusetts General Hospital, where we met with twelve other nervous neophytes to begin
our internships in internal medicine. Within a few days, we developed the friendship
and mutual respect that were to sustain our collaborative effort over four decades.
Our backgrounds were quite different. Joe grew up in small-town South Carolina and graduated
from the young University of Texas Southwestern Medical School in Dallas, where he came under
the spell of Donald W. Seldin, chairman of medicine and intellectual father to generations of
physician-scientists. In stark contrast, Mike grew up in big-city Philadelphia and graduated from
the nation’s oldest medical school, the University of Pennsylvania School of Medicine. We were
drawn together by a shared fascination with clinical medicine and medical science and a desire to
one day make discoveries of significance to both.
In 1968, after two years of residency, we were both accepted for scientific training at the
National Institutes of Health (NIH), where Joe worked in the laboratory of Marshall Nirenberg,
who was soon to receive a Nobel Prize for solving the genetic code. Mike worked with Earl
Stadtman, the biochemist’s biochemist who later received the United States National Medal of
Science. We also had clinical duties. Joe was assigned to care for a pair of siblings (ages 6 and 8) who
were suffering repeated myocardial infarctions due to massively elevated levels of low-density
lipoprotein (LDL) cholesterol in their blood. The diagnosis was homozygous familial hypercholesterolemia (FH), a rare form of a common genetic disease, the pathogenesis of which was
unknown. The two of us discussed these children intensely, and we resolved some day to unlock
the genetic secret behind this striking illness.
In 1970, after two years at the NIH, Joe moved to Seattle to learn medical genetics under the
skillful tutelage of Arno Motulsky. There, Joe spearheaded a classic study of the common Mendelian causes of hyperlipidemia in survivors of myocardial infarction. Mike remained one more year
at the NIH. In 1971, he moved to the University of Texas Southwestern Medical Center, where he
completed a fellowship in gastroenterology and began to study the properties of 3-hydroxy-3methylglutaryl-coenzyme A reductase (HMG-CoA reductase), the rate-controlling enzyme of
cholesterol synthesis that was later shown by Akira Endo to be the target of the statin drugs (Ref.
1; see Ref. 2). Mike’s move to Dallas was strongly advocated by Joe, who enlisted the aid of Seldin.
Mike was willing to try this young medical school. Amazingly, his wife, Alice, a born-again Yankee,
agreed to the move, but only under the condition that they would stay for one or two years at most.
That was four decades ago, and the couple is still happily in residence, having raised two Texan
daughters.
In 1972, Joe returned to Dallas, and we began our collaborative studies of FH. This work led to
the discovery of the LDL receptor, the process of receptor-mediated endocytosis, and the mech-
O
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From the Department of Molecular Genetics, University of Texas Southwestern Medical Center,
Dallas, Texas 75390
REFLECTIONS: Scientific Side Trips
1. Scavenger Receptors and Atherosclerosis
(1978 –1983)
Our discovery of scavenger receptors began with a pathogenic paradox. Patients with homozygous FH lack LDL
JUNE 29, 2012 • VOLUME 287 • NUMBER 27
FIGURE 1. All-or-none difference in receptor-mediated degradation
of 125I-LDL (F) and 125I-acetyl-LDL (E) by human fibroblasts (A) and
mouse peritoneal macrophages (B). This figure was reprinted from
Ref. 18.
receptors; therefore, their cells cannot take up LDL, yet the
same patients accumulate massive amounts of LDL-derived cholesterol in scavenger cells of the body, primarily
macrophages. Moreover, even in subjects with normal
LDL receptors, it is impossible to overload macrophages
with cholesterol by incubation with LDL. When macrophages, or any other cells, begin to accumulate cholesterol,
LDL receptors are reduced by feedback inhibition of
SREBP processing, yet LDL cholesterol accumulates to
high levels in macrophages within atherosclerotic plaques,
converting them into foam cells. Clearly, macrophages
must have an alternate, non-suppressible pathway to take
up LDL.
In 1978, together with Y. K. Ho and Sandip Basu, our
first two postdoctoral fellows, we isolated macrophages
from mouse peritoneal fluid and incubated them with
native LDL (16). As expected, the cells did not accumulate
excess cholesterol because their LDL receptors became
down-regulated (Fig. 1A). We had earlier shown that
acetylation of lysine residues on LDL destroys its ability to
bind to LDL receptors, and we wondered whether the
acetyl-LDL would have acquired the ability to be taken up
by macrophages. To our delight, this was the case (Fig. 1B).
Macrophages expressed a high-affinity receptor that
bound and internalized acetyl-LDL without any downregulation. The excess cholesterol was esterified and
stored in the cytosol as cholesteryl esters, whose concentration was increased by 40-fold in the cells, converting
them to classic foam cells.
Remarkably, the macrophage receptor was not specific
for acetyl-LDL. Other negatively charged macromolecules, including maleyl-LDL, maleyl-albumin, sulfated
polysaccharides (such as fucoidan and dextran sulfate),
and polynucleotides (such as poly(I) and poly(G)) competed for acetyl-LDL uptake. There was some selectivity,
however. Certain negatively charged polymers (such as
poly(C), poly(A), and poly(D-glutamic acid)) did not com-
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anism by which LDL receptors control the level of cholesterol in blood. All of this early work (1972–1985) has been
reviewed elsewhere, most notably in our Nobel lecture,
which was published in Science in 1986 (3). It has also been
detailed in more recent historical reviews (4, 5).
In 1993, our study of the regulation of LDL receptors led to
the discovery of sterol regulatory-element binding proteins
(SREBPs), transcription factors that induce lipid synthesis
and uptake in animal cells (6 –9). The crucial purification was
performed by two postdoctoral fellows, Xiaodong Wang and
Michael Briggs, and the cDNA cloning was accomplished by
Chieko Yokoyama, a postdoctoral fellow, and Xianxin Hua, a
graduate student. The novel feature of SREBPs is that they are
synthesized as intrinsic membrane proteins of the endoplasmic reticulum (ER), and they must be transported to the
Golgi complex, where they are cleaved to send active fragments to the nucleus. We described this transport process
and its feedback regulation in detail and named it regulated
intramembrane proteolysis (RIP). Like receptor-mediated
endocytosis, RIP turned out to be a fundamental biologic
mechanism that is used in more than forty other regulatory
systems, including the proteolytic processing of the developmental protein Notch, the stress protein ATF6, and the amyloid precursor protein, which is cleaved by RIP to form the
pathologic amyloid-␤ peptide (10, 11). Earlier review articles
detail the combined genetic and biochemical approaches
that we used to delineate the transport proteins and proteases
responsible for RIP of SREBPs and to show how these cleaved
transcription factors activate the complete program of cholesterol and fatty acid synthesis in the liver (12–15).
In this Reflections article, we will not discuss LDL receptors, SREBPs, or cholesterol homeostasis. Rather, we focus on
six side projects that we pursued over the years, each affording an adventurous diversion from the central core of our
research and each teaching us a principle useful to science
and medicine. In describing each project, we credit the students and postdoctoral fellows who were most responsible.
However, we do not mean to slight the many others who
made important, even crucial, observations. We value all of
them. Similarly, in citing work from other laboratories, we
focus on the papers that stimulated us or expanded on our
work. This is a very personal list, and we apologize in advance
to the many unnamed scientists who made substantial contributions to each field. This article represents a personal
reflection and not a comprehensive review.
REFLECTIONS: Scientific Side Trips
22420 JOURNAL OF BIOLOGICAL CHEMISTRY
which binds the trace metals that are needed for the lipid
oxidation reaction. Daniel Steinberg and colleagues did
the right experiment and found that oxidized LDL did
indeed bind to the macrophage receptor (24, 25), and Alan
Fogelman and colleagues showed that oxidation of unsaturated fatty acids in LDL generates malondialdehyde,
which forms Schiff bases with lysines, removing the positive charge and mimicking acetylation (26). Although
definitive proof is still lacking, the accumulated evidence
suggests that LDL becomes oxidized in atherosclerotic
plaques, and this causes it to bind to scavenger receptors,
creating the foam cells that are the hallmark of this disease
(27). Antibodies to oxidized LDL may prove to be useful in
blocking macrophage cholesterol accumulation and preventing myocardial infarctions (28).
In 1990, the acetyl-LDL receptor was purified and its
cDNA was cloned by our former postdoctoral fellow
Monty Krieger and his postdoctoral fellows at the Massachusetts Institute of Technology (29). Now called
SR-AI (for scavenger receptor class A, type I), the original scavenger receptor has been joined by a family of
twelve other structurally diverse membrane receptors,
all of which take up acetyl-LDL and/or oxidized LDL.
They also bind and internalize a wide range of other
ligands, including lipid molecules displayed on the surface of bacterial pathogens and modified self-molecules
displayed on apoptotic cells. The extensive literature on
the roles of scavenger receptors in innate immunity,
microbial pathogenesis, and various pathologic processes (in addition to atherosclerosis) has been
described nicely in a series of review articles by Siamon
Gordon and colleagues (30, 31).
2. Prenyltransferases and Choroideremia
(1989 –1996)
We became interested in protein prenylation when we
read the papers of John Glomset and co-workers, who
showed that certain proteins in animal cells possess
hydrophobic prenyl groups, 15-carbon farnesyl or 20-carbon geranylgeranyl attached to cysteines in thioether linkage (32). Prominent farnesylated proteins include Ras proteins, whose activating mutations are among the most
common causes of cancer. Remarkably, Glomset’s findings were made possible by Akira Endo’s discovery of
HMG-CoA reductase inhibitors (1). Thus, a discovery
aimed toward heart disease increased our fundamental
knowledge of cancer. So much for categorical NIH
institutes!
All prenyl groups are derived from mevalonate, which is
produced by HMG-CoA reductase, the rate-controlling
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pete (17). The acetyl-LDL receptor was expressed only on
scavenger cells, including guinea pig Kupffer cells and
human activated monocytes. We speculated that this
receptor is designed to scavenge certain denatured proteins and other macromolecules from the circulation and
that it might be responsible for cholesteryl ester accumulation in human atherosclerotic plaques (18).
Using the scavenger receptor to mediate cholesterol
overload, we next defined the macrophage cholesteryl
ester cycle (19). After uptake, cholesteryl esters of acetylLDL are hydrolyzed in lysosomes, and the free cholesterol is re-esterified in the ER for storage as cholesteryl
ester droplets. The key finding was that the cells
required an external cholesterol acceptor to excrete the
stored cholesterol from the cell. When we added an
external acceptor, such as high-density lipoprotein
(HDL), the stored cholesteryl esters were hydrolyzed by
a cytosolic neutral lipase, and the cholesterol was
released to the acceptor. HDL was not the only acceptor
in blood. Indeed, native LDL could serve as an acceptor,
and so could other plasma proteins, such as thyroglobulin, but surprisingly not albumin. The most potent
acceptors were membranes prepared from red blood
cells, suggesting that red blood cells may be active participants in the shuttling of cholesterol between organs
(20). To our knowledge, this hypothesis has not been
tested, but it may be important in the process that is
now known as reverse cholesterol transport.
We also observed that addition of acetyl-LDL to macrophages stimulated the cells to produce apolipoprotein E
(apoE), which can target lipoproteins for uptake by the
liver (21). This finding presaged the later observations of
Peter Tontonoz, Peter Edwards, and David Mangelsdorf,
who showed that cholesterol loading of macrophages
leads to activation of the nuclear hormone liver X receptor. This receptor activates transcription of a variety of
genes (including those for apoE and ABCA1) that are
designed to release stored cholesterol from macrophages
(22, 23).
We have one regret about the macrophage studies, and
it involves missing oxidized LDL. In thinking about a possible physiologic ligand for the scavenger receptor, we
considered that LDL might undergo oxidation, and some
of the oxidation products may have attached to lysine residues, giving the LDL a negative charge. Accordingly, we
put human LDL in a dialysis bag immersed in a flask filled
with buffer. We bubbled oxygen through the solution
overnight, and the next day, we tested the LDL for uptake
by macrophages. The result was negative. We had
neglected to consider that our buffer contained EDTA,
REFLECTIONS: Scientific Side Trips
JUNE 29, 2012 • VOLUME 287 • NUMBER 27
upstream of the CAAX box. The polylysine sequence gives
K-Ras a 50-fold higher affinity for the farnesyltransferase
compared with H-Ras (41). James Marsters and his colleagues at Genentech designed a series of benzodiazepine
peptidomimetics that were extremely efficient in blocking
farnesylation of H-Ras. Unfortunately, these compounds
were much less effective in blocking farnesylation of
K-Ras. Even more disappointing, we found that when its
farnesylation was blocked, K-Ras became an alternative
substrate for another enzyme that we characterized,
namely geranylgeranyltransferase (now known as
GGTase I) (Ref. 41; see below). Realizing that it would be
difficult to prevent all prenylation of K-Ras, we reluctantly
gave up our quest to cure cancer. Others persisted, but the
clinical results have been disappointing, and currently,
there is no approved farnesyltransferase inhibitor in clinical use.
Our search for a geranylgeranyltransferase began with
the observation by others that geranylgeranyl, not farnesyl,
was added to proteins with CAAX boxes terminating in
leucine. Chief among these are Rac and Rho proteins that
anchor cell membranes to the cytoskeleton. Like Ras proteins, Rho and Rac are GTP-binding proteins. At this
point, Yuval Reiss was joined by Miguel Seabra, a graduate
student from Portugal. Together, they purified the enzyme
that became known as GGTase I. Remarkably, we found
that farnesyltransferase and GGTase I share the same catalytic ␣-subunit. They differ in the ␤-subunit, which confers the specificity for Ras or Rac/Rho proteins, respectively (42).
Miguel Seabra went on to make a discovery that was
even more intriguing. He set out to purify a third prenyltransferase, namely the enzyme that attaches geranylgeranyl to Rab proteins, a large family of GTP-binding proteins
that regulate vesicle fusion reactions. Because Rab proteins do not contain CAAX boxes, Miguel had to use
standard column purification techniques. Surprisingly,
when he applied a partially purified enzyme preparation to
a hydrophobic column, none of the activity was recovered
in the eluate. When this happens, the standard approach is
to mix together the eluate fractions just in case the enzyme
contains two necessary subunits that were separated on
the column. This type of mixing experiment almost never
works. Nevertheless, we urged Miguel to mix the fractions.
All of us were delighted by the result. Indeed, the column
had separated two required components, which we called
Components A and B (43). With this knowledge, Miguel
was able to complete the purification of both components,
always assaying one component in the presence of the
other. Component B contained two polypeptides that
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enzyme in the cholesterol biosynthetic pathway (33).
Glomset’s discovery was triggered by his observation that
blocking HMG-CoA reductase with a statin precluded
cells from entering the S phase of the cell cycle. Adding
cholesterol did not reverse this deficiency, suggesting that
the cells needed another product derived from mevalonate. While blocking production of endogenous mevalonate with a statin, Glomset labeled the cells with [3H]mevalonate and found incorporation into several proteins
(32). Work in yeast and animal cells soon showed that one
set of prenylated proteins always terminates in the amino
acid sequence CAAX, where A stands for aliphatic, and X
can be one of several residues (34, 35). The X residue
determines whether a protein will be modified with a
farnesyl or a geranylgeranyl. Oncogenic Ras proteins are
farnesylated, whereas structural proteins like Rho and Rac
are geranylgeranylated (36). After prenylation, the proteins are cleaved by a protease, so the prenylated cysteine
becomes the COOH-terminal amino acid, and its free
COOH group is then methylated, removing all charges
and allowing the prenyl group to insert into membranes.
Another class of geranylgeranylated proteins called Rab
proteins does not contain a CAAX box. Geranylgeranylation occurs on one or two cysteines near the COOH
terminus.
The findings of Glomset and the yeast geneticists fascinated us because of our longstanding interest in the mevalonate pathway, and we were intrigued by its potential connection to cancer (33). At this point, we were joined by
Yuval Reiss, a postdoctoral fellow from Israel who had
worked on ubiquitination with Aaron Ciechanover and
Avram Hershko and had mastered the technique of affinity chromatography. Yuval made an affinity column with a
CAAX peptide and used it to purify farnesyltransferase
from rat liver. The enzyme was a heterodimer composed
of an ␣-subunit that had the transferase activity and a
␤-subunit that bound to the Ras substrate (37, 38). Yuval’s
enzyme was efficient in farnesylating Ras proteins. Most
importantly, the enzyme could be inhibited by CAAX peptides as short as four residues (37, 39). Others had shown
that farnesylation is essential for the transforming activity
of mutant oncogenic Ras proteins. Together with Guy
James, a postdoctoral fellow, we immediately began a collaboration with Genentech chemists in an effort to design
peptidomimetics that would enter cancer cells and block
Ras prenylation (40).
Ras proteins come in three varieties. H-Ras is the most
heavily studied, but it is not the one that is frequently
mutated in human cancers. That distinction belongs to
K-Ras, which has a polylysine sequence immediately
REFLECTIONS: Scientific Side Trips
22422 JOURNAL OF BIOLOGICAL CHEMISTRY
FIGURE 2. Three heterodimeric prenyltransferases and the REP that
were identified and characterized in our laboratory. GG, geranylgeranyl. This figure was reprinted from Ref. 113.
of choroideremia (51), the first such patient was injected in
Oxford, United Kingdom, in October 2011.1
Our diversion into prenyltransferases did not cure cancer, but it did contribute to our understanding of the enzymology of protein prenylation and may eventually lead to a
cure for a rare form of blindness. If anything, this excursion exemplifies in vivid fashion how curiosity-driven
basic science can have unanticipated positive consequences for medicine.
3. Monocarboxylate Transporters and the Cori
Cycle (1990 –1995)
Monocarboxylate transporters (MCTs) facilitate the
bidirectional movement of short-chain carboxylic acids,
most prominently lactate and pyruvate, into and out of
cells. Their presence had been inferred for many years
based on kinetic studies of monocarboxylate uptake, primarily in red blood cells, but their molecular identities
were unknown.
By pure accident, we isolated the first cDNA for a MCT,
giving birth to a new family that now has fourteen members. The story begins with studies of the uptake of mevalonate, which is not normally a substrate for any MCT. In
1981, Monty Krieger, then a postdoctoral fellow in our
laboratory, developed a method to isolate Chinese hamster ovary (CHO) cells with mutations in the gene for the
LDL receptor (52). After he joined the faculty at Massachusetts Institute of Technology, Krieger and his student
David Kingsley used this approach to isolate a mutant cell
line, designated ldlA-7, which failed to take up LDL
because of a deficiency of LDL receptors (53).
Working with Jerry Faust, formerly a long-term research associate in our laboratory, Krieger attempted to
correct the LDL receptor deficiency by transfecting the
1
M. Seabra, personal communication.
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roughly resembled in size the ␣- and ␤-subunits of farnesyltransferase and GGTase I. Component A was unique
(44).
Miguel Seabra and Doug Andres, a postdoctoral fellow,
then performed a series of kinetic studies that defined the
separate roles of Components A and B. It turned out that
Component B is indeed the catalytic component (45). On
its own, Component B attaches a single geranylgeranyl to a
single Rab protein. In the absence of Component A, the
reaction terminates at that point because the geranylgeranylated Rab remains bound to the enzyme. When Component A is added, it removes the geranylgeranylated Rab
from the enzyme and allows the enzyme to undergo further rounds of catalysis. We thus renamed Component A
as Rab escort protein (REP), later called REP-1. Its function
is to bind the hydrophobic geranylgeranyl group on the
Rab protein, shielding it from water and allowing it to
insert subsequently into the correct vesicular membrane.
CAAX box proteins do not need an escort because the
prenyl group is added while the COOH-terminal residue
retains its hydrophilic negative charge.
We obtained the sequence of six tryptic peptides from
the REP-1 component of Rab GGTase, and when we
blasted them through gene databases, we got an enormous
surprise. The comparison predicted that rat REP-1 is the
orthologue of a human gene that had been implicated in an
X-linked retinal disease called choroideremia, which
causes blindness in young boys (44). We obtained cultured
lymphoblasts from four boys with choroideremia and
found that the cell extracts were severely deficient in
transferring geranylgeranyl to Rab proteins. Activity could
be restored by adding back purified REP-1, but not Component B, confirming that the disease is caused by REP
deficiency (46).
Turning from biochemistry to molecular biology, Doug
Andres, Wen-Ji Chen (postdoctoral fellow), and Scott
Armstrong (M.D./Ph.D. student) cloned cDNAs for the
subunits of all three prenyltransferases (45, 47– 49). Fig. 2
illustrates their subunit composition. When Doug cloned
the cDNA encoding REP-1, he confirmed that the choroideremia gene encoded REP-1 (45). Although REP-1 is
expressed in all mammalian cells, its deficiency causes
only retinal degeneration. As shown by Scott Armstrong
and Frans Cremers (postdoctoral fellow), other cells produce a close relative of REP called REP-2, which substitutes for a deficiency of REP-1 (50). Recently, we became
aware of a gene therapy trial in which a virus encoding
REP-1 is injected into the eyes of boys with this condition.
Following successful preclinical studies in a mouse model
REFLECTIONS: Scientific Side Trips
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FIGURE 3. All-or-none difference in uptake of [3H]mevalonate (left)
and [14C]pyruvate (right) in human MDA-MB-231 breast cancer cells
transfected with cDNAs encoding wild-type pMCT1 or mutant
pMev. This figure was reprinted from Ref. 56.
many cultured cell lines to find one that did not express
wild-type Mev. She identified one such line, a breast cancer line called MDA-MB-231. By transfection, she created
two clones of these breast cells, one expressing wild-type
Mev and the other expressing a similar amount of the
mutant version. She then tested forty-two different radiolabeled compounds to see which of them was taken up
more rapidly by the cells that expressed wild-type Mev but
not the mutant. After many trials, Christine finally found
that [14C]pyruvate met these criteria (Fig. 3) (56). Moreover, pyruvate uptake was blocked by a series of ␣-hydroxycinnamates that had been previously described to
inhibit pyruvate uptake into red blood cells by the presumed MCT (57). We postulated that Mev was indeed the
long-sought MCT, and we named it MCT1. In the met18b-2 cells, MCT1 had undergone a point mutation that
changed the specificity of the transporter from pyruvate to
mevalonate.
Christine prepared an antibody against MCT1. Together
with Ravindra Pathak in Richard Anderson’s laboratory, she
showed that the transporter is expressed highly in erythrocytes, the basolateral epithelium of the kidney, and cardiac
muscle as well as mitochondria-rich skeletal muscle fibers
(56). Surprisingly, MCT1 was not expressed in liver, a finding that led Christine to use a low-stringency hybridization method to screen a hamster liver cDNA library with
her MCT1 probe. The screen yielded a second MCT
(which we called MCT2) that was 60% identical in its
amino acid sequence to MCT1 (58). MCT2 is expressed
predominantly in liver but also in mitochondria-rich skeletal muscle fibers and cardiac muscle. Like MCT1, MCT2
transports both pyruvate and lactate but has a higher affinity for both substrates compared with MCT1.
Christine’s cDNA cloning of MCT1 and MCT2 opened
the floodgates. Currently, fourteen family members are
known (59, 60). The range of substrates transported by
different family members extends from the standard
monocarboxylates, lactate and pyruvate, to aromatic
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cells with a cDNA library prepared from wild-type CHO
cells. They hoped that this approach would lead to the
isolation of the LDL receptor gene. They grew the cells in
a medium called MeLoCo, which was designed to render
cells totally dependent on the LDL receptor for growth.
The medium contained the HMG-CoA reductase inhibitor compactin to block endogenous mevalonate synthesis, thereby forcing the cells to rely on LDL uptake to meet
their cholesterol requirement. The medium also contained a low concentration of mevalonate, sufficient to
supply the small amount of mevalonate needed for trace
non-sterol products like prenylated proteins but not sufficient to meet the cell’s bulk cholesterol need. Lacking LDL
receptors, the ldlA-7 cells cannot grow in this medium.
Faust and Krieger isolated cells (called met-18b-2 cells)
that acquired the ability to grow in MeLoCo, but they had
not taken up any exogenous DNA, nor had they regained
LDL receptors (54). The cells survived because they had
acquired the ability to take up mevalonate 10 – 40 times
more efficiently than the parental cells, and this permitted
mevalonate to satisfy their bulk cholesterol requirements,
even without LDL.
In 1990, we were joined by Christine Kim (now KimGarcia), a fiercely independent M.D./Ph.D. student who
did not want to work on any of the mainline projects in our
laboratory. Christine decided to see whether she could use
an expression cloning strategy to identify the gene responsible for enhanced mevalonate uptake in the met-18b-2
cells. She prepared a cDNA library from the met-18b-2
cells and transfected 500 separate pools of 1000 plasmids
each into dishes of human kidney 293 cells. The cells were
then incubated with [3H]mevalonate, and uptake was
measured. Through multiple reiterations, Christine isolated a cDNA encoding a putative membrane protein with
twelve membrane-spanning regions consistent with being
a membrane transporter but having no sequence homology to any proteins that were in the databases in 1992 (55).
Christine also isolated a cDNA encoding the same protein
from wild-type CHO cells. When the sequences were
compared, we found that the cDNA from the met-18b-2
cells harbored a single point mutation that substituted a
cysteine for a phenylalanine in the tenth membrane-spanning region. We named the protein Mev. Whereas the
Phe-to-Cys Mev mutant endowed cells with the ability to
import mevalonate, the wild-type version did not.
We reasoned that the Phe-to-Cys mutation had altered
the specificity of a membrane transporter so that it
acquired the ability to transport mevalonate. The next task
was to define the natural substrate for the wild-type transporter. This was no easy task. First, Christine screened
REFLECTIONS: Scientific Side Trips
amino acids and thyroid hormones. Through expression
in liver and muscle, the MCT1–MCT4 isoforms facilitate
the Cori cycle, by which glucose is released from the liver
and converted by glycolysis in muscle to lactate, and the
lactate is returned to the liver for resynthesis into glucose.
Thus, the persistence of a talented M.D./Ph.D. student can
advance an important field of physiology, although never
intending to do so.
4. Leptin, Lipodystrophy, and Insulin Resistance
(1997–Present)
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We discovered the first effective treatment for congenital generalized lipodystrophy because of an accident. As
summarized earlier, in 1993, we identified SREBP-1a as an
activator of cholesterol synthesis mediated through sterol
regulatory elements (7). At the same time, Peter Tontonoz
and Bruce Spiegelman identified a related protein that
they called adipocyte determination and differentiation
factor-1 (ADD1) (61). They reported that ADD1 bound to
an E-box in the promoter for fatty acid synthase in adipocytes, enhancing its transcription and increasing the
differentiation of 3T3-L1 preadipocytes in tissue culture.
Because of an artifactual premature termination codon,
the ADD1 sequence did not reveal the unique membranous nature of the protein that we had found in our cDNA
cloning of SREBP-1a. We subsequently determined that
ADD1 was an alternatively spliced form of the SREBP-1
gene that exists in two versions, SREBP-1a and SREBP-1c,
both of which have membrane domains (62). We found
that SREBP-1c activates genes not only for fatty acid synthase, but for all of the other genes necessary for fatty acid
and triglyceride synthesis (13). We also identified
SREBP-2, a third family member encoded by a separate
gene. SREBP-2 preferentially activates all of the genes necessary for cholesterol synthesis (27 enzymes) and cholesterol uptake (LDL receptor). At high levels, it also can activate the fatty acid synthesis genes. After we showed that
the SREBPs must be cleaved in a regulated fashion to
release an active nuclear fragment from the membrane, we
decided to determine the effect that the nuclear form of
SREBP-1c would have on adipose tissue differentiation in
mice. Accordingly, we engineered a transgene encoding
the truncated nuclear form of SREBP-1c that lacks the
membrane domain and thus enters the nucleus in a constitutive unregulated fashion. Transgene expression was
driven by the adipose tissue-specific promoter for aP2, an
adipocyte protein. This is when the accident occurred.
We expected that our transgene would cause an
increase in adipocyte fatty acid synthesis and lead to
enlarged and more numerous adipocytes in the transgenic
mice. In fact, we observed the opposite. The mice lacked
all mature adipocytes in white adipose tissue (63). Although we did not know the reason for this block in differentiation (and we still do not), we realized immediately
that we had created by accident a mouse model of a human
disorder, congenital generalized lipodystrophy (CGL),
also known as Seip syndrome. Subjects with this disorder
have a total lack of white adipose tissue. Inasmuch as they
cannot store triglycerides in adipocytes, these subjects
store them in the liver, which becomes massively enlarged
and steatotic. CGL subjects also manifest hypertriglyceridemia and hyperglycemia caused by severe insulin resistance that elicits marked hyperinsulinemia. Their diabetes
is extraordinarily difficult to control, frequently requiring
injections of hundreds of units of insulin daily. Our lipodystrophic mice (63) shared all of these features with the
human disease (64).
At this point, our postdoctoral fellow Iichiro Shimomura made an important observation. He found that the
adipose tissue remnants in our lipodystrophic mice had a
profound reduction in mRNAs for all of the adipocytespecific genes that were tested, including leptin (63). In
1994, Jeffrey Friedman had made the remarkable discovery that adipose tissue secretes leptin, a peptide hormone
that curbs appetite. Mice with genetic leptin deficiency
(ob/ob mice) ingest massive amounts of food and become
grossly obese, insulin-resistant, and diabetic. In one sense,
our lipodystrophic mice were the opposite of ob/ob mice:
they were pathologically thin instead of being enormously
fat. However, the lipodystrophic mice shared all of the
other characteristics of ob/ob mice, namely massive fatty
liver, severe insulin resistance, and diabetes. Plasma insulin levels in our lipodystrophic mice were 60-fold above
those in wild-type mice, and plasma leptin levels were
barely detectable. Shimomura then asked an impertinent
question: would leptin administration correct the metabolic consequences of lipodystrophy as it does for primary
leptin deficiency? We had little confidence that this experiment would work, for surely adipose tissue must be essential for something other than leptin production.
Accordingly, Shimomura administered leptin to our
lipodystrophic mice for twelve days through an osmotic
minipump. The results were dramatic (65). Even after this
short period, plasma insulin fell to normal, plasma glucose
was normalized, and the fatty liver resolved completely
(Fig. 4). The mice were still lipodystrophic; they had no
discernable white fat, yet their insulin resistance and diabetes were reversed by administration of only a single
product from adipose tissue, leptin. The hormone
decreased food intake by 16% in the lipodystrophic mice,
REFLECTIONS: Scientific Side Trips
but the metabolic resolution could not be explained solely
by reduced food intake. In the absence of leptin treatment,
an even greater forced reduction in food intake (30%)
failed to reverse the diabetes and fatty liver (65).
Before we published these observations in Nature, we
contacted our UT Southwestern colleague Abhimanyu
Garg, an endocrinologist who had collected several
patients with CGL. At that time, The Rockefeller University had licensed leptin to Amgen. Accordingly, in August
1999, we sent a preprint of our Nature paper to Amgen
and proposed an exploratory clinical trial of leptin therapy
in Garg’s patients with lipodystrophy. Six months later, in
February 2000, while negotiations with Amgen were still
under way, a Brief Communication appeared in Nature
that threatened to abort the trial. The Communication,
from Marc Reitman and co-workers in the Diabetes
Branch of the National Institute of Diabetes and Digestive
and Kidney Diseases (NIDDK), reported studies of
another mouse model of lipodystrophy in which leptin
administration had only a negligible ameliorating effect on
blood glucose and insulin (66). They challenged our conclusion by stating, “Evidence from humans and mice supports the conclusion that leptin deficiency cannot completely explain the diabetic phenotype of generalized
lipodystrophy.” They also stated that “leptin deficiency…is
neither the sole cause nor the principal cause of insulin
resistance in severe forms of this disease.”
The publication of the Brief Communication from the
NIH cast a pall on the rationale for a clinical trial of leptin
in CGL. We remained convinced by our own data in mice,
and we believed that patients with CGL deserved a chance
at a new treatment. Fortunately, Simeon Taylor and Phillip
Gorden, two senior scientists in the Diabetes Branch, kept
their minds open. They and their junior associate Elif Oral
agreed to participate in the clinical trial and to enroll the
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FIGURE 4. Histological sections of liver (hematoxylin and eosin
stain) from lipodystrophic mice treated for twelve days with either
PBS (A) or recombinant leptin (B). Vacuolated areas represent triglyceride droplets (magnification, ⫻200). This figure was reprinted from Ref.
65.
CGL patients who were being followed at the NIH.
Together, the Garg group and the NIH group completed
the study, and the dramatic results were published in the
New England Journal of Medicine in February 2002 (67).
Nine female patients were treated with leptin for four
months. All showed a dramatic drop in blood sugar and
insulin, a dramatic reduction in liver and plasma triglycerides, and a dramatic reduction in liver size. All of the diabetic patients showed a profound reduction in the need for
glucose-lowering medications. One teenage girl, who had
required a massive insulin dose of 3000 units/day prior to
therapy, required no glucose-lowering agent when treated
with leptin. We now know that the effects of leptin persist
with time. In the most recent published report, more than
100 patients with severe forms of lipodystrophy have been
treated with leptin with persistent relief of diabetes, fatty
liver, and hypertriglyceridemia (68).
The findings in mouse and human lipodystrophy have
major implications for our notion of the role of adipose
tissue in metabolism. Although adipose tissue secretes
hormones in addition to leptin, including adiponectin and
resistin, the absence of adipose tissue is compatible with
relatively normal glucose homeostasis as long as a single
hormone, leptin, is replaced.
We were intrigued with the concordant findings of
severe insulin resistance with hyperglycemia, severe
hypertriglyceridemia, and severe hepatic steatosis in leptin-deficient lipodystrophic and ob/ob mice. In a milder
form, this constellation of abnormalities is also characteristic of humans with the metabolic syndrome and type 2
diabetes. In all of these states, the hyperglycemia is attributed to insulin resistance, primarily in liver, muscle, and
adipose tissue. Insulin resistance in liver was long known
to trigger an increase in hepatic gluconeogenesis due to a
failure of insulin to down-regulate genes such as phosphoenolpyruvate carboxykinase and glucose-6-phosphatase, which are crucial for the synthesis and secretion of
glucose by the liver. On the other hand, our laboratory (69)
and those of Bruce Spiegelman (70) and Pascal Ferré (71)
had shown that insulin activates fatty acid synthesis in
rodent liver by increasing the amount of nuclear SREBP1c. In the insulin-resistant liver, how can insulin continue
to stimulate SREBP-1c production while losing its ability
to suppress gluconeogenesis?
Together with Shimomura, we focused on the paradox
of simultaneous insulin resistance and sensitivity in the
livers of lipodystrophic and ob/ob mice. We showed that
this condition correlated with a down-regulation of IRS-2,
one of the two proteins that mediates the signaling action
of insulin in liver (72). We pointed out that the mixture of
REFLECTIONS: Scientific Side Trips
5. Niemann-Pick Type C Proteins and the
Hydrophobic Handoff (2006 –Present)
Our foray into Niemann-Pick type C (NPC) disease
began with the work of a gifted M.D./Ph.D. student, Rodney Infante. Rodney obtained his undergraduate degree in
accounting. After graduation, he worked in the business
world before enrolling in our medical school as a straight
M.D. student. After his first year, he spent a summer working in our laboratory, and he remained for the next 5.5
years. For his thesis project, Rodney accepted the formidable challenge of purifying a membrane-bound oxysterol-binding protein.
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Oxysterols such as 25-hydroxycholesterol are potent
blockers of the ER-to-Golgi transport of SREBPs, and thus,
they are potent suppressors of cholesterol synthesis. In
fact, oxysterols are much more potent than cholesterol.
The inhibitory actions of oxysterols and cholesterol are
totally dependent on Insigs, polytopic membrane proteins
in the ER that trap the Scap-SREBP complex and prevent
its incorporation into vesicles that exit the ER (76, 77).
Arun Radhakrishnan, a postdoctoral fellow, showed that
cholesterol acts by binding to Scap, triggering a conformational change that causes Scap to bind to Insigs (78). However, Arun was unable to show that Scap binds oxysterols.
Therefore, we postulated that ER membranes must contain an as-yet-unidentified protein that binds oxysterols
and facilitates the binding of Scap to Insigs. Rodney’s task
was to identify and purify this putative oxysterol-binding
protein.
Rodney was a neophyte when it came to protein purification, so we encouraged him to enroll in the two-week
protein purification course at Cold Spring Harbor. There
he learned a great deal. It is a real technical challenge to
purify a hydrophobic membrane protein that binds a
hydrophobic ligand like an oxysterol. The ligand and the
binding protein must both be dissolved in detergents,
which create innumerable opportunities for artifacts.
Nevertheless, Rodney persisted. Within a year, he had
devised six chromatographic steps to obtain from rabbit
liver a protein preparation that was purified by 14,000fold. To identify the oxysterol-binding protein, he crosslinked his preparation with a derivative of 25-[3H]hydroxycholesterol. After SDS gel electrophoresis, he
excised the labeled band and identified the protein by mass
spectroscopy. The identified protein was encoded by the
gene that is defective in a human genetic disease of cholesterol metabolism called NPC disease (79).
NPC disease is an autosomal recessive lysosomal storage disease in which cholesterol and other lipids accumulate in lysosomes, particularly in liver, spleen, lung, and
brain (80). Affected individuals appear normal in infancy,
but they exhibit progressive hepatic and neural degeneration, usually dying before the teenage years. NPC disease
was initially lumped together with Niemann Pick type A
(NPA) disease, which is caused by a deficiency of lysosomal acid sphingomyelinase. However, in the 1980s,
Peter Pentchev and Roscoe Brady at the NIH realized that
NPC was distinct from NPA. NPC cells had normal acid
sphingomyelinase activity, yet they accumulated large
amounts of cholesterol and sphingomyelin when grown in
tissue culture.
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insulin resistance and insulin sensitivity (which we now
call “selective insulin resistance”) (73) leads to a vicious
cycle: the insulin-resistance component of the cycle causes
the liver to secrete excess glucose into plasma, stimulating
the pancreas to produce more insulin, which fails to suppress gluconeogenesis; the insulin-sensitive component of
the cycle allows the increased plasma insulin to continue
to activate SREBP-1c and stimulate fatty acid synthesis,
resulting in fatty liver and hypertriglyceridemia. As originally formulated by our late and lamented colleague Denis
McGarry, hypertriglyceridemia causes excess fatty acids
to be delivered to peripheral tissues, thereby exacerbating
their insulin resistance and leading to further increases in
plasma glucose and insulin and even greater production of
triglycerides in liver (74). This vicious cycle characterizes
not only lipodystrophy and leptin deficiency, but also the
common form of type 2 diabetes associated with obesity,
leptin resistance, and the metabolic syndrome.
In recent years, our laboratory has continued to study
the hepatic paradox of selective insulin resistance. With
postdoctoral fellow Shijie (Chris) Li, we showed that insulin induction of SREBP-1c mRNA in hepatocytes and in
livers of living rats is blocked by rapamycin, the inhibitor
of mammalian target of rapamycin (mTOR) (75). Rapamycin does not inhibit insulin suppression of gluconeogenesis, indicating that the two insulin signaling pathways
diverge proximal to mTOR.
Currently, there is intense interest in many laboratories
to identify the pathway by which insulin increases nuclear
SREBP-1c mRNA and thereby increases fatty acid synthesis. If this limb of the vicious cycle can be interrupted, it is
possible that some of the negative consequences of type 2
diabetes can be ameliorated. Thus, the accidental discovery of a mouse model of lipodystrophy may some day lead
to effective treatments for a major epidemic that is engulfing the industrialized world.
REFLECTIONS: Scientific Side Trips
JUNE 29, 2012 • VOLUME 287 • NUMBER 27
of his in vitro assay. Rather, sterols bind to the NH2-terminal end of NPC1, which is a sequence of 240 amino acids
that projects into the lysosome lumen. When expressed as
a truncated protein without the membrane domain, the
NH2-terminal domain (NTD) is secreted into the culture
medium. After purification, it binds cholesterol and
25-hydroxycholesterol with saturation kinetics (79).
Cross-competition studies with related sterols indicated
that the binding site recognizes the sterol nucleus, including the 3␤-hydroxyl on the A-ring, but it is indifferent to
hydrophilic substitutions on the isooctyl side chain on the
D-ring (88). The protein does not recognize epicholesterol, which differs from cholesterol only in the orientation of the 3-hydroxyl group, which is in the ␣-position in
epicholesterol. Rodney also prepared recombinant NPC2,
and he found that the soluble protein recognized the
opposite end of the sterol (88). NPC2 bound epicholesterol, but it did not bind 25-hydroxycholesterol, suggesting that it recognizes the hydrophobic isooctyl side chain
on the D-ring, but not the 3␤-hydroxyl on the A-ring.
At the same time that Rodney was performing his comparative binding studies, Lobel and Ann Stock published
the structure of NPC2 bound to cholesteryl sulfate as
determined by x-ray crystallography (89). Their structure
supported the idea that NPC2 binds cholesterol by recognizing the sterol rings and the isooctyl side chain, but the
3␤-hydroxyl does not interact with the protein. Indeed,
NPC2 bound cholesteryl sulfate even with the bulky sulfate attached to the 3␤-hydroxyl. Rodney showed that
cholesteryl sulfate does not bind to NPC1, further heightening the differences between NPC1 and NPC2 (88).
Mouse knock-in experiments confirmed that the cholesterol-binding activity of NPC1(NTD) is essential for
NPC function in living animals. In preparation for these
studies, Rodney and another M.D./Ph.D. student, Michael
Wang, performed alanine scanning mutagenesis of
NPC1(NTD) to identify residues that were crucial for sterol binding. They identified two adjacent amino acids
(Pro-202 and Phe-203) that, when changed together to
alanine, eliminated cholesterol binding in vitro. Another
graduate student, Lina Abi-Mosleh, showed that fulllength NPC1 containing these adjacent alanine substitutions failed to complement the defect in NPC1-deficient
CHO cells (90). Building upon these in vitro observations,
our colleague Guosheng Liang, together with postdoctoral
fellow Xuefen Xie, produced knock-in mice homozygous
for the P202A/F203A mutation in the npc1 gene and
showed that they exhibit a clinical and pathologic syndrome indistinguishable from that in npc1nih/nih mice, the
classic model for NPC1 deficiency (91).
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Our early work had shown that cholesterol and sphingomyelin are delivered to lysosomes by the receptor-mediated uptake of LDL. After liberation from LDL, the cholesterol leaves the lysosome and is transported to the ER
and plasma membrane. In 1985, Pentchev and Brady
showed, indeed, that the source of excess lysosomal cholesterol in NPC cells was the LDL of the culture medium
(81). When NPC cells were incubated with LDL, the LDLderived cholesterol failed to leave the lysosomes. As a
result, there was a delay in the suppression of cholesterol
synthesis and the activation of cholesterol re-esterification. The gene encoding the NPC protein was isolated by
positional cloning in 1997 (82). Its sequence predicted a
protein of 1278 amino acids with thirteen transmembrane
helices. It was named NPC1. The protein was localized to
lysosomal membranes but had never been purified, and
there was no information about its biochemical properties. Peter Lobel and colleagues showed that a minority of
NPC patients owe their disease to defects in another protein that they named NPC2 (83). NPC2 (then called HE1)
had been identified earlier as a cholesterol-binding protein
in epididymal fluid. Lobel showed that NPC2, a 132-amino
acid soluble protein, is also targeted to lysosomes, where it
resides in the lumenal fluid. The protein was missing in
patients with NPC disease who did not harbor a mutation
in NPC1. Defects in NPC1 or NPC2 produce the same
clinical phenotype, suggesting that they may act sequentially in transporting cholesterol out of lysosomes (84, 85).
Rodney’s discovery of NPC1 as an oxysterol-binding
protein intrigued us. We were even more enthralled when
he showed that the protein also binds cholesterol (79). By
this time, Radhakrishnan had shown that the oxysterol
sensor for SREBP processing is not an unknown protein;
indeed, it is Insig itself, which binds oxysterols but not
cholesterol (86). Rodney’s NPC1 protein was not involved
in feedback regulation of SREBP processing, but it was
even more interesting because it was crucial for cholesterol export from lysosomes, a process that was totally
mysterious.
Rodney then expressed deletion mutants of NPC1 so as
to identify the domain that bound sterols. Here, we got
another surprise. Among the thirteen transmembrane
helices of NPC1, five show sequence resemblance to membrane-spanning sequences in Scap, HMG-CoA reductase,
and certain other proteins that are regulated by sterols.
We had noticed this ⬃170-amino acid domain originally
in Scap, naming it the sterol-sensing domain (87). We
expected that sterols would bind to this domain of NPC1.
However, Rodney showed that sterols do not bind to the
membrane domain of NPC1, at least under the conditions
REFLECTIONS: Scientific Side Trips
22428 JOURNAL OF BIOLOGICAL CHEMISTRY
FIGURE 5. Model for the hydrophobic handoff of cholesterol from
NPC2 to NPC1, a step in the exit of LDL-derived cholesterol from
lysosomes. This figure was reprinted from Ref. 93.
patches on both proteins met each other, and the bound
cholesterol was in the same plane in the two proteins (94).
On the basis of this alignment, we proposed a model that
we called the hydrophobic handoff (Fig. 5).
The hydrophobic handoff model (93, 94) postulates that
soluble NPC2 binds cholesterol immediately after acid
lipase releases it from the cholesteryl esters of LDL. NPC2
carries the cholesterol to the lysosomal membrane, where
it binds to the NTD of NPC1, which projects through the
glycocalyx, the thick carbohydrate lining that prevents lysosomal proteins from direct contact with the limiting
membrane (95). Binding of NPC2 induces a conformational change in the NTD of NPC1 that moves aside the
obstructing helices and opens the sterol-binding pocket.
Once this entrance is opened, cholesterol slides directly
from NPC2 to NPC1. After the handoff, the NPC2 dissociates, and the surface helices on the NTD close again,
locking the cholesterol in the binding pocket. The great
virtue of this handoff is that it shields the cholesterol from
water. From the moment that cholesterol leaves LDL, it is
always bound to a protein, and it never has the opportunity
to crystallize, which would be a catastrophic event in the
lysosome. This model has been expanded recently by
Suzanne Pfeffer and her colleague, who have shown that
NPC2 binds to a lumenal loop of NPC1 that is adjacent to
the NTD (96). This binding may facilitate the transfer of
cholesterol from NPC2 to the NTD of NPC1.
If the hydrophobic handoff model turns out to be correct, it still leaves a major problem: how does the cholesterol move from the NTD of NPC1 to enter the membrane
and emerge on the other side for transport to the ER and
plasma membrane? Here, it is likely that the thirteen
membrane-spanning helices of NPC1 play a role, but how
they function is a complete mystery. In fact, we know very
little about how cholesterol is transported between any
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Rodney’s biochemical observations that NPC2 and
NPC1 were both cholesterol-binding proteins suggested
that the two proteins might transfer cholesterol between
themselves (92). In his initial studies, Rodney had observed
that cholesterol bound very slowly to NPC1(NTD) at 4 °C.
Moreover, after binding cholesterol, NPC1(NTD) slowly
transferred the bound sterol to phospholipid liposomes.
However, when NPC2 was added, NPC1(NTD) bound
cholesterol many times more rapidly, and it also transferred cholesterol much more rapidly to liposomes. A naturally occurring NPC2 mutant that does not bind cholesterol failed to accelerate association and dissociation of
cholesterol from NPC1(NTD). We hypothesized that
NPC2 acts as a cholesterol shuttle, either delivering cholesterol to NPC1 or removing it. To test this hypothesis,
Rodney designed a direct transfer assay in which he first
isolated NPC2 with [3H]cholesterol bound and then incubated this complex with NPC1(NTD). Whereas the NTD
bound free cholesterol extremely slowly, binding was
accelerated by orders of magnitude when the sterol was
delivered directly by NPC2 (92).
At this point, our studies were advanced immensely
through a collaboration with Hyock Kwon, a postdoctoral
fellow in the laboratory of our colleague Johann Deisenhofer. Together, Hyock and Rodney crystallized the NTD
of NPC1 with and without bound cholesterol (93). The
structures confirmed the conclusions drawn from the
binding studies. Cholesterol was buried in a deep pocket
within the NTD, which formed tight contacts with the
sterol ring and particularly with the 3␤-hydroxyl group.
The isooctyl side chain extended through a small tunnel to
the surface, projecting away from the binding pocket.
Importantly, the entry to the cholesterol-binding pocket
was occluded by three ␣-helices that must have moved
aside to admit the cholesterol and then closed behind it to
lock the sterol in place.
Rodney and Michael Wang performed additional alanine scanning mutagenesis of NPC1(NTD), and they identified a small patch of residues on the surface of the NTD
that were not required for the NTD to slowly bind free
cholesterol but were essential for rapid acceptance of cholesterol transferred from NPC2 (93). We called these
“transfer” mutants, distinct from the “binding” mutants.
Later, Michael and Rodney made a similar alanine scanning mutagenesis study of NPC2 and identified a similar
patch of residues on the surface of NPC2 that were
required to transfer cholesterol to NPC1(NTD) (94).
Working together with Hyock and using the crystal structure published by Lobel and Stock, Michael and Rodney
were able to align the two proteins such that the transfer
REFLECTIONS: Scientific Side Trips
organelles in a cell. This may be a topic for a future excursion from the beaten path.
6. Ghrelin O-Acyltransferase and Surviving
Starvation (2006 –Present)
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We were attracted to ghrelin as soon as we found out
that it is the only peptide hormone that bears a covalently
attached fatty acid. Even more unique is its distinction as
the only known eukaryotic protein that bears an eightcarbon fatty acid (octanoate). Lipid modifications of proteins have fascinated us ever since our studies of protein
farnesylation, and we were eager to uncover the enzyme
that attaches octanoate.
Ghrelin was discovered in 1999 by Masayasu Kojima,
Kenji Kangawa, and associates in Osaka as a stomach-derived hormone that releases growth hormone from pituitary cells (97). They characterized ghrelin as a 28-amino
acid peptide with an octanoate attached in ester linkage to
a serine at the third position from the NH2 terminus.
Ghrelin’s activity in releasing growth hormone from the
pituitary and hypothalamus is strictly dependent on the
octanoate moiety.
Ghrelin came to prominence when its plasma concentration was shown to rise dramatically before meals and to
decline immediately after eating in humans and rodents
(98, 99). Moreover, injection of excess ghrelin increased
food intake in humans as well as rodents (100 –102). These
findings led to the hypothesis that ghrelin is responsible
for hunger before meals and satiation after eating. The
validity of the hunger hypothesis came into question when
the gene for ghrelin or its receptor was eliminated in mice
through germ line recombination (103–105). These deficient animals grew normally and showed only inconsistent
and mild decreases in food intake and body weight.
Intrigued by ghrelin’s unique octanoate modification,
we decided to identify and purify the putative acyltransferase that attaches this fatty acid. In 2006, we assigned the
task to an enterprising graduate student, Jing Yang. We
knew that biochemical purification would be difficult
because ghrelin is produced in fewer than 1% of gastric
mucosal cells (102). Nevertheless, Jing devised an assay to
measure [3H]octanoate transfer from [3H]octanoyl-CoA
to desacyl proghrelin. He did purify an enzyme that catalyzed this reaction, but it turned to be N-myristoyltransferase, which was clearly not the relevant enzyme. At this
point, we became aware of the bioinformatic work of Kay
Hofmann, who had defined a family of membrane-bound
enzymes that transfer fatty acids to serines or threonines
on lipids such as cholesterol or on proteins such as Wingless, a secreted protein in the Wnt pathway (106). Hof-
mann’s data indicated that the mammalian genome
encodes sixteen such enzymes, which he termed membrane-bound O-acyltransferases (MBOATs). The substrates for eight of the sixteen presumed MBOATs were
unknown when we began our search.
Jing cloned cDNA copies of fifteen MBOATs by PCR of
mRNAs from mouse stomach. To assay these putative
enzymes for ghrelin O-acyltransferase activity in intact
cells, Jing first identified an insulin-secreting rat cell line
called INS-1, which is capable of cleaving transfected preproghrelin to ghrelin but is not capable of acylating the
ghrelin (107). He then established a reverse-phase chromatographic assay to separate acylated ghrelin from
desacyl ghrelin. He transfected the INS-1 cells with a plasmid encoding preproghrelin plus a plasmid encoding one
of the fifteen MBOATs. He extracted the ghrelin from the
cells and used his reverse-phase column to determine
whether it was acylated. All results were negative. At this
point, we had no assurance that the INS-1 cells could acylate ghrelin even if the enzyme was supplied. Perhaps the
enzyme required the assistance of another protein that
was missing from the cells. It was also possible that the
presumed ghrelin O-acyltransferase was not a member of
the MBOAT family at all.
Jing pointed out that he had been unable to amplify one
of the sixteen MBOATs from the mouse stomach mRNA.
He proposed to make new attempts to isolate this last one.
Frankly, we were very skeptical. Having failed with the first
fifteen MBOATs, what was the probability that the sixteenth would succeed? Nevertheless, Jing persisted. He
found that the problem was that the mouse genome project contained an incorrect annotation of the exon at the
5⬘-end of the gene, so Jing’s primers could not have
worked. To circumvent this problem, Jing pieced together
the putative cDNA by ligating synthetic oligonucleotides
that corresponded to the available partial mouse cDNA
sequence. He used 5⬘-rapid amplification of cDNA ends
and nested PCRs to extend his cDNA to the 5⬘-end of the
mRNA (107). This was an enormous amount of work simply to test whether the sixteenth MBOAT would be the
sought-after one. Almost miraculously, these experiments
worked. When transfected into INS-1 cells, Jing’s synthetic sixteenth MBOAT cDNA produced octanoylated
ghrelin, as did the authentic cDNA that he subsequently
cloned. After our paper had been reviewed and accepted
for publication in Cell, Gutierrez et al. (108) from Eli Lilly
& Company submitted a paper to the Proceedings of the
National Academy of Sciences of the United States of
America in which they identified the same MBOAT that
we had described as a mediator of ghrelin octanoylation.
REFLECTIONS: Scientific Side Trips
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FIGURE 6. Progressive hypoglycemia in Goatⴚ/ⴚ mice subjected to
60% calorie restriction for seven days. *, p ⬍ 0.01; **, p ⬍ 0.001. This
figure was reprinted from Ref. 110.
of food. Moreover, when fasted, they showed the same
signs of hunger as did wild-type mice. We were not surprised by these results because they are the same as
observed in knock-out mice lacking ghrelin or the ghrelin
receptor.
Persisting in the idea that ghrelin must play a role in
appetite regulation, we decided to determine whether
wild-type mice would outlive Goat⫺/⫺ mice when placed
in conditions in which food was limited. We planned to
place one wild-type mouse and one Goat⫺/⫺ mouse in the
same cage but provide an amount of food that was sufficient only for one. However, prior to performing this
experiment, we decided to see what would happen if we
kept the wild-type mouse and the Goat⫺/⫺ mouse in separate cages but provided each with only 40% of the amount
of food that they normally consumed in one day. We called
this “60% calorie restriction,” and the results were dramatic. Faced with 60% calorie restriction, wild-type and
Goat⫺/⫺ mice became progressively hungrier. The mice
were fed each day at 6 p.m. After a few days, all of the mice,
Goat⫺/⫺ as well as wild-type, consumed their entire food
allotment within 1 h. Over the first four days, wild-type
and Goat⫺/⫺ mice lost 30% of their body weight and more
than 80% of their body fat. We measured blood sugar levels
at 5:30 p.m., 23 h after the mice had completed their last
meal. Despite their fat loss, wild-type mice were able to
maintain blood sugar levels in the viable range, always
above 40 mg/dl. In dramatic contrast, by day 7, Goat⫺/⫺
mice developed severe hypoglycemia with blood sugar levels reaching as low as 12 mg/dl (Fig. 6). At this point, the
Goat⫺/⫺ mice appeared moribund, and they were killed
(110).
We also measured plasma hormone levels at 5:30 p.m.
Wild-type mice showed progressive daily increases in
ghrelin, desacyl ghrelin, and growth hormone. By day 7,
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They studied a line of thyroid carcinoma cells that
secreted octanoylated ghrelin, and they screened siRNAs
targeting candidate MBOATs for their ability to block
ghrelin acylation.
The sixteenth MBOAT in Jing’s studies and the one
identified by Gutierrez et al. corresponded to MBOAT-4
in Hofmann’s original list. We renamed this enzyme
GOAT, which stands for ghrelin O-acyltransferase (107).
The tissue distribution of GOAT mRNA coincided with
the distribution of ghrelin, being found largely in the stomach and upper small intestine. We established an in vitro
assay to measure the transfer of octanoate from octanoylCoA to proghrelin (109). Ghrelin corresponds to the first
twenty-eight amino acids of proghrelin, which is generated after the protein is inserted into ER membranes and
the signal sequence has been cleaved. After transport to
the Golgi, proghrelin is cleaved by a furin-like protease to
generate the mature 28-amino acid ghrelin. GOAT
attaches octanoate to Ser-3 of proghrelin, which corresponds to the site originally identified in ghrelin by Kojima
and Kangawa. We found that GOAT did not require a
full-length protein for activity. Indeed, the enzyme transferred octanoate to a peptide corresponding to the first
five amino acids of proghrelin. GOAT was sensitive to end
product inhibition by octanoylated peptides. The most
potent inhibitor (Ki ⫽ 0.2 ␮M) was a pentapeptide in which
diaminopropionic acid was substituted for Ser-3, and the
octanoate was attached to this residue in amide linkage
rather than the less stable ester bond (107). These studies
raised the possibility of designing inhibitory peptidomimetics to inhibit GOAT in vivo provided a relevant clinical
indication could be identified.
In an effort to understand the true physiologic or pathologic role of ghrelin and to confirm the role of GOAT in
producing the hormone, we collaborated with scientists at
Regeneron Pharmaceuticals, Inc., to eliminate the Goat
gene in mice by germ line recombination. In Dallas, these
experiments were conducted by Tong-Jin Zhao, a skilled
postdoctoral fellow from Tsinghua University, with expert
help from our colleague Guosheng Liang. Because there is
some ambiguity in nomenclature, in the following discussion, we use the term “ghrelin” to denote the octanoylated
28-amino acid mature form of the hormone that circulates
in blood. “Desacyl ghrelin” denotes the unacylated form,
which is also found in the circulation. GOAT-deficient
mice had no circulating ghrelin, confirming the indispensable role of GOAT in mediating the acylation reaction
(110). Circulating levels of desacyl ghrelin were actually
elevated in the GOAT knock-out mice. The GOAT-deficient mice grew normally and consumed normal amounts
REFLECTIONS: Scientific Side Trips
JUNE 29, 2012 • VOLUME 287 • NUMBER 27
appears to be due primarily to a limitation in available
substrates and energy rather than to a problem with the
production of gluconeogenic enzymes. A crucial question
is how ghrelin-induced growth hormone can maintain the
levels of gluconeogenic substrates under a condition in
which there are no fat stores and where there is prolonged
food deprivation. Solving this problem will shed light on a
hitherto unknown function of growth hormone as well as
ghrelin.
Starvation is a frequent bottleneck in the evolution of
animals, and nature clearly has selected for mechanisms to
maintain blood sugar at levels sufficient to keep the heart
beating and the brain functioning. Our evidence indicates
that ghrelin is an essential agent in blood sugar maintenance. Depleted fat stores and food deprivation are
observed in humans suffering from chronic starvation or
from anorexia nervosa. In both cases, plasma levels of
ghrelin and growth hormone are elevated, as they are in
calorie-depleted fasted mice (112). We therefore believe
that ghrelin helps to maintain blood sugar by stimulating
growth hormone secretion in humans. Our excursion into
GOAT and ghrelin began with the persistence and skill of
Jing Yang and was extended by the meticulous animal
experiments initiated by Tong-Jin Zhao and Guosheng
Liang.
Concluding Remarks
The six excursions described in this Reflections article
were made possible by three extraordinary privileges that
we have enjoyed over four decades. The first is a steady
parade of talented students and postdoctoral fellows from
around the world who came to a scientifically youthful
place like Dallas, Texas, braved difficult challenges, and
solved problems that often seemed unsolvable. In this
Reflections article, we cite the research of students and
postdoctoral fellows from ten different countries in addition to the United States. Moreover, our continuing work
on cholesterol metabolism has been aided by students and
postdoctoral fellows from seventeen additional countries
(a total of twenty-seven countries). We sincerely hope that
the United States continues to be a magnet for the brightest talent from around the world.
The second privilege is generous and sustained philanthropic support from enlightened citizens of Dallas who
believed in us and our work. We particularly thank the late
Erik Jonsson, Ross Perot, and Peter O’Donnell, Jr., three
giants of Dallas. Each of them made an enormous difference not only to us, but to our medical school and to our
city. When we embarked on each of our six excursions, we
had no preliminary data of the type required by review
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the two hormone levels were more than 4-fold above those
in ad libitum fed wild-type mice. In Goat⫺/⫺ mice, ghrelin
was undetectable, desacyl ghrelin rose to wild-type levels,
and growth hormone barely increased. Hypoglycemia in
Goat⫺/⫺ mice was prevented when the animals received
constant infusions of either ghrelin or growth hormone via
an osmotic minipump beginning three days prior to instituting calorie restriction (110).
In a subsequent study, Tong-Jin, together with M.D./
Ph.D. student Robert Li and postdoctoral fellow Daniel
Sherbet, studied the diurnal pattern of blood sugar in the
calorie-restricted mice (111). Even after seven days, the
Goat⫺/⫺ and wild-type mice both had normal blood sugar
levels of 60 mg/dl when measured at 9 a.m., 15 h after
finishing their last meal. Thereafter, wild-type mice maintained stable blood sugar levels in this range throughout
the day of fasting. In Goat⫺/⫺ mice, by 2 p.m., the blood
sugar began to decline significantly, and it reached its
nadir at 5:30 p.m., 23 h after the last meal. In both strains,
glucose rose immediately after eating the next meal. The
same end-of-day hypoglycemia was observed in calorierestricted ghrelin knock-out mice, indicating that hypoglycemia in the Goat⫺/⫺ mice was indeed caused by ghrelin deficiency. The above studies indicated that this
hypoglycemia required two events: 1) chronic calorie
restriction to deplete body fat stores nearly completely and
2) a prolonged fast of 23 h in the fat-depleted state.
In their most recent experiments, Tong-Jin and Sherbet
infused [3H]glucose into wild-type and Goat⫺/⫺ mice and
demonstrated that the end-of-day decrease in blood sugar
is caused by a 60% reduction in glucose production (111).
The decreased glucose production could not be attributed
to a decrease in the production of gluconeogenic enzymes
at the mRNA level. Indeed, the Goat⫺/⫺ mice had nearly
undetectable levels of plasma insulin and markedly elevated levels of glucagon, as would be expected in the hypoglycemic state. Moreover, the liver responded to the glucagon by generating marked elevations in mRNAs
encoding phosphoenolpyruvate carboxykinase and glucose-6-phosphatase, two glucagon-induced enzymes of
glucose production (110, 111). Plasma levels of lactate and
pyruvate, two important substrates for gluconeogenesis,
were reduced by 50% in the hypoglycemic Goat⫺/⫺ mice.
Moreover, the hypoglycemia could be reversed by injections of lactate, pyruvate, or alanine, all of which can be
converted to glucose. Hypoglycemia was also corrected by
infusing octanoate, a medium-chain fatty acid that cannot
be converted to glucose but can provide energy and reducing equivalents that are necessary for gluconeogenesis
(111). Thus, hypoglycemia in calorie-restricted mice
REFLECTIONS: Scientific Side Trips
Address correspondence to: mike.brown@utsouthwestern.edu or joe.
goldstein@utsouthwestern.edu.
FIGURE 7. Inevitable changes in Brown (right) and Goldstein (left)
that have occurred over our forty-six years of friendship and partnership. A, 1966: interns in medicine at the Massachusetts General Hospital. B, 1974: budding young scientists at the time of discovery of the
LDL receptor. C, 1985: newly announced recipients of the Nobel Prize in
Physiology or Medicine. D, 2012: senior scientists.
committees of the NIH. No committee would have funded
experiments that were supported only by the following
outrageous hypotheses.
1) Macrophages have receptors that scavenge a variety
of abnormal macromolecules, one of which is responsible
for cholesterol accumulation in atherosclerotic plaques. 2)
Blindness in young boys with choroideremia is caused by a
failure to attach geranylgeranyl groups to Rab proteins. 3)
Studies of mutant CHO cells that take up mevalonate
might lead to the molecular identification of the longsought MCT. 4) Studies of an artificial model of lipodystrophy in mice might lead to the first effective treatment
for the disease in humans and provide fundamental
insights into selective insulin resistance in livers of
humans with type 2 diabetes. 5) The polytopic membrane
protein NPC1 contains a cholesterol-binding site, not in
its membrane domain but in its soluble NH2-terminal
extension that accepts cholesterol from NPC2, thus pro-
22432 JOURNAL OF BIOLOGICAL CHEMISTRY
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In each case, we used our philanthropic underpinning
to initiate these high-risk studies, none of which would
have been possible without such support. In contrast, we
used our generously funded long-term NIH program project grant to support our work on the regulation of cholesterol metabolism, which over the years became mainstream and thus approvable by NIH review panels.
The third privilege that we have enjoyed is working
with brilliant colleagues at the University of Texas
Southwestern Medical School in Dallas. Under the
inspired leadership of our former president, Kern Wildenthal, our school recruited many of the finest scientists whose work spans the spectrum of modern biomedical research. With some, we have engaged in
formal collaborations. With others, we have benefitted
from endless hours of excited discussions on all manner
of topics. Their insights shine through our work.
Fig. 7 documents the inevitable changes that have
occurred over our forty-six years of friendship and
partnership.
REFLECTIONS: Scientific Side Trips
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