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I
FRONTIERS IN THE NUTRITION SCIENCES
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MOLECULAR BIOLOGY AND NUTRITION RESEARCH
Richard W. Hanson, Maria Hatzoglou, Mary M. McGrane,
Fritz M. Rottman, and Thomas Wagner
Metabolic research is the foundation of the science of
nutrition, and progress in one area is linked to the
vitality of the other. Metabolic research has not
prospered over the past decade, when compared with
research in fields such as genetics, due in part to a
limitation in the techniques applied to metabolic
problems. For too long metabolic research has focused on
redefining problems of regulation without developing new
methods powerful enough to provide definitive answers to
these problems. The result has been a general demise of
metabolism as an area of modern biology. With its
research base contracting, nutritional science has become
a more descriptive and less vital scientific field.
The revolution in molecular biology has brought new
opportunities and a fresh challenge for metabolic
research. To date, research in molecular biology has
focused on the characterization of specific genes and on
the processes that alter their expression. Many genes
have been sequenced and their promoter-regulatory regions
have been delineated, and various mutations involved in
disease processes have been described. However, the
ability to isolate and characterize a gene of interest is
the first step in a process that can involve the
modification and expression of that gene in cells and
animals. It is here that the opportunities for the
direct application of molecular biology to the field of
metabolic research has the potential for its greatest
impact. This brief review outlines some of the
techniques in this rapidly evolving field and speculates
on their application to metabolic research.
3
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ISOLATION AND CHARACTERIZATION OF GENES OF METABOLIC
INTEREST
The first area in which molecular biology will have an
impact on metabolic research is the isolation and
characterization of genes that code for proteins of
metabolic interest. To date, understanding of the
regulation of a key metabolic enzyme, such as the
pyruvate dehydrogenase complex, phosphofructokinase, or
P-enolpyruvate carboxykinase (PEPCK), ha's been limited by
a total lack of knowledge of the primary o'r
three-dimensional structures of these proteins or the
configuration of the ligand-binding domains, which are
critical to their functioning. As the list of genes of
this type that have been'sequenced' "rows, there will soon
be a complete series of structures on which to base
experimental' approaches to understanding the regulation
of key enzymes. This initial phase of characterization
of metabolically important genes is well under way, and
it is safe to predict that within the next 5 years, most
of the major 'regulatory enzymes in important metabolic
pathways such as glycolysis and gluconeogenesis will have
been cloned, sequenced, and studied further.
The availability of complementary DNA (cDNA) clones for
specific regulatory enzymes will permit their
overexpression in bacteria and purification of the
protein. Coupling of these techniques with site-directed
mutagenesis of the DNA will permit a detailed analysis of
structure-activity relationships for individual enzymes.
This level of analysis requires knowledge of the complete
crystal structure of the protein to accurately assign
specific amino acids within the protein. 'At this point,
the determination of the crystal structure of a protein
is the rate-limiting step in studying'its function by
site-directed mutagenesis. This fact is reflected in the
current demand for (end ' shortage of)''X-ray '
crystallographers. It is ironic that it is now easier to
isolate, clone, and sequence a gene than it is to
characterize it by established physical methods. '
An excellent example of the great power of the combined
techniques discussed above is the work of Robert
Fletterick and colleagues on the characterization of
glycogen phosphorylase (Sprang et al., 1987; S. Sprang,
E. Goldsmith, and R. Fletterick, unpublished data). This
dimeric enzyme, which is present in muscle and liver
4
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tissues (Krebs and Fisher, 1956), has been purified and
its crystal structure determined (Sprang et al., 1987,
,~nn7,hl i chap ~! The It's is regulated by two
parallel mechanisms. The first of these involves its
phosphorylation by a glycogen, phosphorylase kinase,
which converts the enzyme from the inactive lo-form to the
active a-form (Krebs and Fisher, 1956~. Glycogen
phosphorylase kinase is, in turn, phosphorylated (and
activated) by a cyclic AMP (cAMP)-dependent protein
kinase. This mechanism of liver glycogen phosphorylase
regulation is directly linked to the level of blood
glucose via alterations in the concentration of glucagon.
The second type of regulation involves control of the
activity of the enzyme by a series of intracellular
intermediates, including AMP, ATE, and
glucose-6-phosphate. These two mechanisms of regulation
working together ensure a coordinated response to changes
in energy metabolism and to the carbohydrate status of
the organism. Such control is central to metabolic
regulation and forms the basis of the nutritional
response to carbohydrate intake.
_ --~ ~ ~ J · ~ 4~_ AWLS ~ ~ ~
Glycogen phosphorylase b2 the unphosphorylated form of
glycogen phosphorylase' binds AMP at a site on one of the
subunits, which in turn promotes further binding of this
ligand to a site on the second subunit. Glycogen
phosphorylase b is inactive in the absence of AMP and is
80% as active as Glycogen phosphorylase a when AMP is
bound to the enzyme (Green and Cori, 1943; Morgan and
Parmeggiani, 1964~. Both ATP and glucose-6-phosphate
inhibit glycogen phosphorylase b by competing with AMP
and stabilizing the catalytically inactive conformation
of the enzyme. An understanding of the nature of the
complex interaction between these regulatory molecules
has been limited by the absence of a detailed
structure-function analysis of glycogen phosphorylase.
In a recent publication, Sprang et al. (1987) presented
a detailed analysis of the structure of the glycogen
phosphorylase nucleotide activation switch. That study
elegantly demonstrated the power of the combined
techniques of molecular biology and protein chemistry
when they are applied ~ ' ~ ~
interest. Since the c DNA for the enzyme was cloned and
the primary sequence of the phosphorylase was deduced
from the nucleotide sequence, a detailed map of the amino
acids at the enzyme's active site was determined. From ~
co a regulatory enzyme or metabolic
5
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the crystal structure of the protein, Sprang and
coworkers showed that the subunit interface of glycogen
phosphorylase is composed of a catalytic and a regulatory
domain (Figure 1~. The catalytic site of the enzyme was
B i\
/ Dome, n
('
~ .:
-
\
r
At
. . .
. .
.
....
. ..
...
.....
...
......
,
.. ....
/
_~/Regulatory ~
W ~moi>>
. ~
FIGURE 1 Schematic representation of changes in the
dimeric glycogen phosphorylase a molecule.
Abbreviations: S. L~gand-binding sites for glucose and
glucose-l-Pi G. glycogen and oligosaccharides; and N. AMP
and ATP. SOURCE: From Sprang et al. (1987), with
permission of the authors.
mapped to a crevice in the protein between the two
domains, while the regulatory domain was found at the
interface of the two subunits. The amino acid residues
involved in nucleotide binding were identified, and their
relative affinities for AMP and ATE were determined. A
reaction mechanism based on the structure of both the
6
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regulatory and catalytic sites on glycogen phosphorylase
can thus be derived, and the confirmational changes that
occur in the enzyme after ligand binding can be
described.
A detailed presentation of these studies is beyond the
scope of this review; however, the work of Sprang et al.
(1987) is an example of the refined analysis of enzyme
regulation that will provide a molecular basis for the
next generation of metabolic studies. Because of this
work, it is now possible to discuss the regulation of
glycogen phosphorylase in terms of its molecular
interactions with regulatory molecules and to alter the
metabolic function by site-directed mutagenesis based on
an understanding of the reaction mechanism. Information
of this type for other enzymes can be expected to
increase rapidly as the sequencing and crystal structure
analyses become available. ~ knowledge of the enzyme
structure at this level also permits the rational design
of modified proteins for metabolic studies involving gene
transfer to cells and animals. This is discussed below
in more detail.
THE INTRODUCTION OF GENES INTO CELLS AND ANIMALS
Construction of Chimeric Genes
Many structural genes that code for proteins of
metabolic interest have been isolated and characterized
(Goodridge and Hanson, 1986), including genes that code
for key regulatory enzymes in metabolic pathways,
hormones such as growth hormone, and insulin and a
variety of receptors, to name only a few genes of
interest. The number of proteins that have been isolated
and characterized is growing rapidly and is likely,
ultimately, to include virtually all of the important
proteins involved in metabolic processes.
At the same time, the control regions within the
5-flanking gene sequences are being isolated and
studied. It is this promoter-regulatory region of a gene
that controls its expression. Specific sequences
contained in this complex region of a gene interact with
transcriptional regulatory factors' which are often
intermediates in hormone action, to- control the
tissue-specific expression of the gene in animals. It is
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now possible to construct genes that contain a chimeric
promoter-regulatory region that contains, for example,
selected ho`mone-responsive elements, a promoter element
with appropriate strength, and a tissue-specific element
that directs expression of the gene to a tissue of
interest. - This chimeric promoter-regulatory region can
these ligated to a segment of DNA coding for a
structural gene of interest and can be introduced into
cells or animals.
As the techniques~for stably introducing genes into
cells and animals improve, it should be possible to
reproducibly target genes for selected tissues in animals
by using these chimeric genes. This is a new tool with
great potential for metabolic studies, since it will
permit, for the first time, a modification of an
enzymatic step in a complex metabolic pathway without the
use of inhibitors or other compounds that have a broad
It also provides
the potential to correct metabolic defects in humans, if
the technology can be perfected to ensure a predicted
site of integration into the human genome, as well as a
normal level of expression of the newly introduced gene.
We review here some aspects of this field and use as an
example our own studies with the PEPCK gene, since its
promoter-regulatory region has proven useful in driving
the expression of a variety of structural genes in a
regulated and tissue-specific manner. We also review the
techniques currently being used to introduce genes into
cells and animals in order to demonstrate both the.
limitations of and potential for these techniques.
spectrum of action in cells or animals.
Properties of a Regulated Promoter
The selection of an appropriate promoter-regulatory
region for use with a linked structural gene in metabolic
studies depends on the tissue in which the gene is to be
expressed and the type and level of regulation of gene
expression that is required. The promoter-regulatory
region of the gene coding for the cytosolic form of PEPCK
(GTP) (EC 4.1.1.32) contains a highly regulated promoter
sequence, with regulatory elements for cAMP (Short et
al., 1986; Wynshaw-Boris et al., 1984, 1986),
glucocorticoids (Wynshaw-Boris et al., 1984, 1986),
insulin (Magnuson et al., 1987; McGrane et al., 1988)
and thyroid hormone (M. Hatzoglou, W. Lamers, A.
8
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Wyushaw-Boris, and R.W. Hanson, unpublished
observations), all within 550 base pairs (bp) of flanking
DNA at the 5' end of the gene. This gene also has a very
high level of transcription (3,500 parts per million
after administration of cAMP) (tamers et al., 1982;
Meisner et al., 1983) in transgenic mice and
tissue-specific sequences are present in the promoter
that directs gene expression, to the liver and kidney
(McGrane et al., 1988~. The messenger RNA (mRNA) for
PEPCK has a half-life of 30 minutes (Granner et al..
1983 Til~hman et al
~ ~ ,
_ __, ~__= ~.^ ~ ., 1974), so that the level of mRNA
for the enzyme is largely dependent on alterations in the
transcription rate of the gene. Hormones such as
glucagon (acting via cAMP), glucocorticoids, or insulin
have a very marked and rapid effect on PEPCK gene
transcription.
a-
_ _
The administration of Bt2cAMP to an animal causes an 8-
to 10-fold induction in the rate of transcription of the
Gene within 20 minutes (tamers et al., 19821. This
effect can be blocked in cells if insulin is administered
together with the cAMP (Granner et al., 1983;
Wynshaw-Boris et al., 1986b). The PEPCK promoter thus
has many advantages for use as a vehicle- to drive the
expression of linked genes in animal tissues; it is
expressed in a tissue-specific manner and is regulated
acutely by hormones, and its promoter strength is great
enough to ensure sufficiently high levels of gene
product.
~,
~. ~. .
_,_ ~
The various hormone response elements in the promoter
have been identified by gene transfection experiments in
which chimeric genes containing segments of the
promoter-regulatory region of the PEPCK gene were ligated
to the structural gene for a selectable marker, such as
the amino-3'-glycosyl phosphotransferase gene, which
makes cells resistant to the cytotoxic compound, G418, or
the Herpes virus thymidine kinase (TK) gene (Short et
al., 1986; Wynshaw-Boris et al., 1984,-1986b). Figure 2
shows the locations of sequences in the PEPCK promoter
that confer sensitivity to cAMP and glucocorticoids in
the chimeric PEPCK-TK gene. Two cAMP response elements
have been mapped by DNA-protein footprinting to the
region between positions -82 to -90 and -135 to -142
(Roesler et al., 1989~. The entire region of DNA between
positions -61 and -416 can act as a hormonally sensitive
enhancer and can regulate expression when it is
9
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a, O
a'
~ ~ o so
a, - ~ to ~ o
- ~Jo
it ~o ~ ~ ~
/
/ ~v o ~ ~ no
/ ~no =^ ~ ~
<. ~ / ~ ~ ~ ~ ~ ~
~ / ~ ~ ~ ~ o ~ ~
~ u o ° L,~ R ~ -a 9 ~ _ ~ .
~ oJo~o~ ~ >~ i ;
\ ~o ~ on ~ o
- of ~
\ ~=m == ~ ·
\ mO o Cot ~
o \ ~Ed e 3 ~ m" :~
~\ } ~ ~ ~ "m
\ .~, ~ ~ al to Cal
\ :~' Ha o
c ~ ~ ~ .-
\ ~ ~ ~ o
~.. ~04 8 8 ~ e e
a) al a, a,
u' ~ ~ a, ~
10
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introduced into the chimeric gene in either
transcriptional orientation or at a distance from the
start site of transcription of the gene (Figure 3;
Wyushaw-Boris et al., 1986b).
REGULAT10N
cAMP DEX
-6s0
~1
-548
+1 +73
,-.
-
-355 +1 +73
-~74 +1 +73
-109 +1~73
+
+
_ __ ~
-68 +1 +73
-109 +1
-109 -62/-l09+l
+ +
~_ _ _ _ _ _ ~+ +
-62 -10~-l09+l
-548 -10~-K9+'
-416 -61/ - 109 +'
,.
-61 -416/-109 +1
+
___c , . /, + +
-109 +1 +1800/-416 -61
___~, ,* + +
-109 +1 +1800~-61 -416
-S48 +73i-650 +1
-S48/-650
~1
FIGURE 3 Functional analysis of the PEPCK
promoter-regulatory region for cAMP and glucocorticoid
regulatory elements. Rat hepatoma cells deficient in TK
(cont. next page)
11
+
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cAMP, glucocorticoids, and thyroid hormone (Hatzoglou et
al., unpublished observations). The relative level of
KNA synthesized from these newly introduced genes was an
order~of magnitude lower than the level of PEPCK mRNA
synthesized by the endogenous gene (Figure 7~. The
ability to direct the expression of a chimeric gene into
the tissues of an animal relatively late in development,
however, holds promise as a technique of potential
usefulness for broader metabolic studies aimed at
introducing modified genes of interest into animals to
alter various metabolic and nutritional parameters.
INTO -28
,.lj~
:~e
·~o
710 bp ~ ~ -
37S bp - `
fETAL INJECTION
, . . .
t
1
In ~ z
z ~ _
~ _ ~
11
1
: ~
1 . .,
t . - -:
. _L_ _ _ . .; ~ ; --- t.;c
i .'." i,'.
~-_
8C ~ provirus pLJ-PCKneo
- I I I I I Bgl Ir ~* 32 p - 5'- end lobe led DNA probe
111111 viral LTR RNa
ITIL
~ 2 3 4 S ~ 2 3
Fl
- 3
~1
-.
0710bp
~ 375 bp
PCK- neo RNA
FIGURE 7 Expression of the neo gene introduced into
FTO-2B hepatoma cells and in rat liver by retroviral
(cont. next page)
21
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(fig. 7 caption cont.)
infection and its induction by Bt2cAMP. The infectious
retrovirus pLJPCKneo was introduced into FTO-2B hepatoma
cells and into the peritoneal cavities of 19 fetal rats
in utero. The virus had a titer of 8 x 107
G418-resistant colonies of NIH 3T3 cells per ml of
virus-containing medium. Fetal rats were injected with
100 pi of virus of the same titer at 19 days of fetal
life and were then killed at 3 months after birth. A
quantitative S1 nuclease analysis of neo mRNA in FTO-2B
cells after treatment with Bt2cAMP is presented on the
left. The band present at 375 bp is a protected fragment
corresponding to the predicted size of the neo gene probe
used in these studies and indicates the mRNA transcript
from the PEPCK promoter of the infected retrovirus. The
diagram under the panels shows the orientation and size
of the hybridization probe used, as well as the size of
the predicted fragments protected from S1 nuclease. The
710-bp band represents mRNA transcribed from the 5' LTR
of the vector. Bt2cAMP (0.1 ~M) was added to the medium,
and RNA was extracted from the cells 3 hours later.
Insulin (10-8 unitsj was Added at the same time as the
Bt2cAMP or 2 hours later (next to the last lane in the
left panel). Rats were injected later with Bt2cAMP 3
hours prior to death. Their livers were removed' and the
RNA was\extracted and analyzed for neo sequences. The
two panels on the right are the result of two separate
exposures of the bands protected during S1 nucleate
mapping of RNA from the liver. The three lanes on the
far right were exposed to X-ray film for 48 hours i and
the five bands on the left were exposed for 24 hours.
The relative intensities of the bands are proportional to
the concentration of neo mRNA present in the cells.
SOURCE: Data from Hatzoglou et al. (unpublished
observations).
Tissue-Specific Expression and Dietary Regulation of a
Chimeric PEPCK-bGH Gene Introduced into the Germ Line of
Mice
A series of transgenic mice were produced by the
microinJection of a chimeric PEPCK-bGH gene into the male
pronucleus of fertilized mouse eggs by the technique
outlined above and as described by Wagner et al. (1981~.
22
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The microinjection experiments were performed by Drs.
June Yun and Thomas Wagner (Edison Animal Biotechnology
Center, Ohio University, Athens, Ohio). The
promoter-regulatory region of the PEPCK gene contained
450 bp of 5'-flanking sequence, which included the cAMP
and glucocartic regulatory elements. The concentration
of bGH in the serum of animals that contained the gene
stably integrated into their genome and ranged from a low
of 5 to more than 2,300 ng/ml of serum (McGrane et al.,
1988~. Mice with high levels of bGH grew at twice the
rate of their littermates but were normal in all other
respects.
A detailed Northern blot analysis of animal tissues
indicated that the chimeric PEPCK-bGH gene was expressed
in only the liver and the kidney. This mirrors the
expression of the endogenous PEPCK gene, which is also
highly expressed in liver and kidney and only marginally
in other animal tissues (Hanson and Garbers, 1972~. The
level of bGH mRNA in the livers of these transgenic
animals was regulated by hormones and diet in a manner
similar to that of the endogenous gene. When starved
animals were fed a diet high in carbohydrate for 1 week,
there was a 95% decrease in the concentration of bGH in
the serum of the mice (Figure 8~. This suggests that the
expression of the PEPCK-bGH chimeric gene in these
animals is sensitive to the insulin released after
glucose ingestion. When the mice were refed a diet high
in protein but devoid of glucose, the levels of bGH in
serum were increased 30-fold after 1 week on the diet.
This inductive effect of a high protein and
carbohydrate-free diet on the levels of hepatic PEPCK is
part of the response of the animal to the need for
enhanced gluconeogenesis. The transgenic mice also
responded to the administration of Bt2cAMP by increasing
the level of bGH in their serum two- to threefold in 90
minutes (see Figure 8~.
23
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0.5
0.4
3
0.3
0.2
0.1
A
B
(24 hr)
_ (1 week)
(1 week)
. _
1
1
Bt2AMP
(90 min)
starved High High
CHO Protein
No CHO
3
2
1
FIGURE 8 Regulation of the bGH concentration in the
blood of transgenic mice by diet and Bt2cAMP
administration. (A) A transgenic mouse expressing 300 ng
of bGH per ml was starved for 24 hours and then fed a
diet composed of 82% sucrose, 12.2% casein, 0.3%
dl-methionine, 4% cottonseed oil, 2% brewer's yeast, and
a 1% mineral mix plus vitamins for one week. Blood was
drawn from the tail vein; the animal was then fed a diet
containing 64% casein, ~
22% a-cell nutritive fiber, 11%
And 1 & mineral mix with
vegetable oil, 2% brewer's yeast
vitamins (synthetic diets were from Nutritional
Biochemical Corporation) for 1 week. The concentration
of bGH in the blood was determined. (B) A mouse
expressing 1.4 fig of bGH was injected with Bt2cAMP and
theophylline (both 30 mg/kg of body weight) at three
consecutive intervals. At 90 minutes the mouse was bled
from the tail vein and the concentration of bGH was
measured (McGrane et al., 1988~.
24
-
3:
c,
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The acute responsiveness of the PEPCK promoter-
regulatory region to induction by diet and hormones and
its tissue-specific expression in the liver and kidney
makes it an ideal tool for targeting the expression of
various structural genes of interest to these tissues.
It is also possible to modulate the level of expression
of the structural gene over a broad range by altering the
carbohydrate content of the diet fed to transgenic
animals. Since the gene for the cytosolic form of PEPCK
is normally not expressed until birth (Ballard and
Hanson, 1967), the developing fetus is not exposed to a
high level of protein that would result from expression
of the structural gene. This has clear advantages with
hormones such as bGH, which have the potential of
interfering with the normal development of the fetus.
have noted a normal developmental pattern for the
transgenic animals that we have studied to date. It
should also be possible to use a segment of the PEPCK
promoter, when it is linked to a structural gene of
interest that contains its own core promoter but that
lacks homologous tissue-specific elements, to direct the
expression of this chimeric gene to the liver and kidney.
POSSIBLE APPROACHES TO GENETICALLY BASED ALTERATIONS IN
METABOLIC PROCESSES
There are many metabolic models that can be used to
test the potential of the techniques described above. An
example is the role of the mitochondrial isozyme of PEPCK
in the regulation of hepatic gluconeogenesis. Since the
discovery of PEPCK in chicken liver mitochondria by
(Utter and Kurahaski, 1954), as well as its cytosolic
isozyme in the livers of a variety of species (Nordic and
Lardy, 1963), the metabolic roles of the two forms of
PEPCK have remained unresolved (Hanson and Mehlman,
1976~. This has been compounded by the fact that species
vary in the relative amounts of the two forms of the
enzyme that are present in their livers. In birds, for
example, 100% of the hepatic PEPCK is the mitochondrial
isozyme, while in rodent species such as rats and mice,
90% of the enzyme is the cytosolic enzyme. The majority
of species studied to date, including humans, have equal
amounts of the two forms of PEPCK (Hanson and Garbers,
1972~. These enzymes are distinct, but related proteins
that have approximately 60% sequence identity and are
coded for by different nuclear genes (S.M. Weldon, S.
25
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Savon, W. C. Merrick, and R.W. Hanson, unpublished
observations).
The only gluconeogenic tissue known to have a single
form of PEPCK is the liver of the chicken, which contains
only the mitochondrial isozyme (Watford et al., 1981~.
This distribution of the enzyme creates metabolic
restrictions on the ability of the chicken to synthesize
hepatic glucose. The lack of cytosolic PEPCK in the
avian liver has been cited as a reason for the low rates
of gluconeogenesis noted with oxidized substrates such as
pyruvate or alanine in this tissue (Williamson, 1976~.
Lactate conversion to pyruvate in the cytosol provides
the NADH required by the glyceraldehyde-3-phosphate
dehydrogenase reaction during gluconeogenesis. In the
liver of the chicken, the restricted synthesis of
P-enolpyruvate in the mitochondria necessitates a
continuous supply of NADH from oxidation-reduction
reactions in the cytosol. However, for gluconeogenesis
from pyruvate or alanine, the transfer of reducing
equivalents, as well as carbon, from the mitochondria is
required. In tissues that possess a cytosolic form of
PEPCK, this is accomplished by the transport of malate
into the cytosol, which is then converted to oxalacetate,
generating NADH (Williamson, 1976~. In tissues that lack
cytosolic PEPCK, the carbon for gluconeogenes~s muse
leave the mitochondria as P-enolpyruvate without the
transfer of reducinz equivalents.
.
In chickens glucose
_ ,
synthes~s ~n the liver is restricted to the Cori cycle,
whereas net gluconeogenesis from amino acids occurs in
the kidney, a tissue that contains both isozymic forms of
PEPCK (Watford et al., 1981~. Thus, birds recycle
lactate from the muscle and red blood cells to the liver
for gluconeogenesis
It should be possible to test the metabolic role of the
two forms of PEPCK directly in the hepatic cells isolated
from chickens, in which the gene for the cytosolic form
of the enzyme has been introduced via an infectious
retrovirus by the techniques described above. The gene
for the cystosolic form of PEPCK from chickens has been
isolated, and a full-length cDNA is available (Hod et
al., 1984 a,b). Since the gene can be inserted into the
liver via infection of fertilized eggs with the
replication-competent retrovirus described above, the
levels of gene expression can be measured both by
determining the concentration of PEPCK mRNA present in
26
OCR for page 27
the liver and by assaying for enzyme activity in the
cytosol present in the liver. The effects of this
genetic modification of the cellular localization of the
rate-limiting enzyme in hepatic gluconeogenesis should
have dramatic effects on glucose homeostasis in chickens.
Therefore, a method is available to test the predictions
of the metabolic model discussed above.
Alternatively, it should be possible to target the
mitochondrial isozyme of PEPCK to the hepatic
mitochondria in mice. This would involve the
construction of a chimeric gene consisting of the
promoter-regulatory region of the gene for the cytosolic
form of PEPCK, which contains both hormone regulatory
elements as well as sequences directing the
tissue-specific expression of the gene to the liver and
kidney, linked to the structural gene for the
mitochondrial form of PEPCK from chickens, which also
contains the signal sequences necessary to direct the
protein to the mitochondria of murine liver. A cDNA
coding for this isozyme has recently been isolated
(Weldon et al., unpublished observations).
Alternatively, the availability of a hepatoma cell line
with minimum deviation that can grow in a glucose-free
medium, such as the Fao cell line isolated and
characterized by Weiss and colleagues (Deschatrette and
Weiss, 1974), will be an invaluable tool in these types
of studies, since it permits infection with the murine
retroviral vectors already developed and has been shown
to effectively introduce functional and regulatable genes
into other hepatoma cells. Since the cells are able to
synthesize glucose from the gluconeogenic precursors
contained in the culture medium, their growth in the
absence of added glucose should be directly dependent on
the efficiency of gluconeogenesis. Finally, once these
genetically modified cells are constructed and
characterized, they are a permanent resource for other
investigators who are interested in related metabolic
problems.
The scenario presented above represents only one of
many metabolic questions that might be approached by
using the techniques outlined here. We anticipate rapid
growth in this field as the number of genes that are
isolated and characterized increases. It should be
possible, for example, to introduce receptors for
hormones such as insulin into the membranes of cells in
27
OCR for page 28
which these receptors are either present in low number or
missing. Such an approach has already been accomplished
with the human placental insulin receptor, which has been
introduced into rodent CHO cells (which have a low number
of insulin receptors) and has been shown to respond to
added insulin by greatly increasing the transport of
glucose into the cells (Ellis et al., 1986~. However
the metabolic consequences of altering the uptake of
glucose in these cells was not considered. There
currently is a unique opportunity to extend the
usefulness of studies of this type into the area of
metabolism and nutrition.
THE NEED FOR NEW EDUCATIONAL PROGRAMS COMBINING
METABOLISM AND MOLECULAR BIOLOGY
The important advances that have occurred in molecular
and cellular biology over the past 15 years have great
potential for metabolic and nutritional research. A core
technology centered around the ability to introduce
specific genes into cells and animals in a directed and
regulatable fashion is now developing. This technology
will be generic in its usefulness in-biology and medicine
and will extend from the possible correction of genetic
defects to the genetic patterning of cells and animals
for metabolic studies. The field of nutrition has a vast
potential as a research area in which this technology
will find application. As discussed above, it will soon
be possible to alter specific steps in a complex
metabolic pathway in intact animals to determine the
animals' responses to individual dietary components or to
target the expression of hormone receptors to tissues
that are normally unresponsive to a specific hormone.
For the potential of these new approaches to be fully
realized in the nutritional sciences, a new generation of
investigators who are familiar with molecular biology, as
well as with metabolism and nutrition, must be trained.
Without these individuals, the potential application of
many of these techniques will be needlessly delayed.
Unfortunately, among graduate students who are attracted
to the fast pace and high visibility of molecular
genetics, there is a diminished interest in metabolism
and nutrition as a research area. There is also a gulf
between faculty in the nutritional sciences and those in
molecular biology in most universities. Two cultures
28
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have arisen that have different languages, different
traditions, and different approaches to science.
Unfortunately, these differences continue to grow. The
information contained in this volume based on the
symposium "Frontiers in the Nutrition Sciences" could
make an important and timely contribution to the
development of a program for education in nutrition. We
hope that the nutritional sciences will share in the
excitement of the new advances in molecular and cellular
biology by participating in a revitalization of metabolic
research, the basis for all nutritional science.
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