Chapter 36. Nutritional Regulation of Gene Expression

Figure 36.2. Basic aspects of gene expression and protein synthesis. Nutritional regulation of genes can occur directly or through hormones or other mediators that

activate signaling systems.



Transcription rates for individual genes can be altered by various mechanisms and represent the major point of gene regulation. Factors influencing the binding of

RNA polymerase to DNA, specifically the promoter region of the gene (the 5'-flanking region) and other sequences in close proximity, control transcription. These

factors (transcription factors) are proteins with domains that recognize a specific nucleotide sequence (response element) located in the promoter. Response

elements are usually short sequences found within the first few hundred nucleotides upstream from the start site of the structural gene. There are exceptions,

however. As more promoter sequence information is obtained for upstream (>1000 bases) sequences, it is clear that distant response elements may, through various

mechanisms or interactions, influence basal-level transcription rates. Specific transcription factors form dimers or other complex combinations through protein-protein

interactions among regulatory proteins or molecules to influence transcription. These aspects of transcription have been reviewed in detail ( 7).

The direct interaction of response elements and transcription factors with nutrients has been documented. Examples include the sterol response element for

sterol-regulated genes ( 9), the calcitriol receptor-response elements for vitamin D–regulated genes ( 10), the retinoic acid receptor-response element combinations for

vitamin A–regulated genes (11), fatty acids and some of their metabolites ( 11A, 11B) and the metal response elements for zinc-regulated genes ( 12) and associated

transcription factors. The human genome contains approximately 100,000 genes, of which 10% may be transcriptionally active at any one time ( 13). Consequently,

many unidentified response elements may be under nutrient control. In addition, cytokines and hormones act by altering gene expression, usually transcription ( 8),

and response elements for these factors may exist at other locations within a gene's promoter that are also influenced by a specific nutrient or dietary pattern.

Furthermore, the abundance of some transcription factors in cells can be regulated by physiologic processes that may be influenced by diet.

It is unlikely that a specific gene is regulated by a single response element or even multiple copies of a response element in a promoter. An example of the likely

complexity is the phosphoenolpyruvate carboxykinase promoter which, within the first 1 kb of upstream sequence (5' from the transcription start site), has five

response-element sequences (cAMP, glucocorticoid, insulin, peroxisome proliferator–activated receptor, and thyroid hormone) and may interact with at least 10

transcription factors (14).



IDENTIFICATION OF GENES REGULATED BY INDIVIDUAL NUTRIENTS OR DIETARY PATTERNS

The following section describes some techniques and approaches of particular interest to investigators studying the effect of nutrition on gene expression. Examples

of these techniques from the contemporary literature concentrate on the results of experiments with animals or human subjects fed diets that produce metabolic

changes.

Polyacrylamide Electrophoresis of Proteins and Immunoblotting

Extremely valuable information can be obtained by measuring specific gene products (proteins) related to altered phenotypic expression. For such purposes, proteins

are most frequently separated by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) using any of the innumerable variations of the PAGE

format (15). To identify the proteins of interest, proteins are electroblotted to nitrocellulose (or another membrane) from the polyacrylamide gel for specific times to

ensure that the proteins are retained on the nitrocellulose. The protein(s) of interest is usually detected immunologically by a process referred to as western blotting.

Immunodetection methods allow investigators to examine a specific protein among the thousands of tissue proteins transferred to the membrane. The spectrum of

antibodies available for these applications (western blot) from commercial sources is substantial. Alternatively, many investigators working on newly identified or rare

proteins use antibodies produced to chemically synthesized peptides corresponding to a specific region of the intact protein that is likely to be highly antigenic. These

peptides usually correspond to 20 to 30 amino acids of the protein. Frequently, the amino acid sequence is deduced from the cDNAs of genes for which the gene

product has not yet been isolated and sequenced ( 16). This approach allows examination of the nutritional regulation of proteins encoded by newly identified genes.

Furthermore, the sensitivity of some immunodetection methods allows examination of proteins present in low abundance.

Small proteins or peptides used to elicit antibodies are conjugated to keyhole limpet hemocyanin or another protein to enhance antigenicity. Larger proteins are most

frequently used without conjugation. Antibodies are most frequently raised in rabbits, but goats and sheep are often used ( 15). Immunization of chickens and recovery

of antibodies from egg yolk is gaining in popularity ( 17). Mammalian polyclonal antibodies are usually purified by protein A or G chromatography to obtain the IgG

fraction. Frequently, these are further selected by chromatography using a support to which the protein or peptide antigen has been coupled to yield a monospecific

IgG population.

Monoclonal antibodies are also widely used in nutrition-related studies on gene expression. The availability of hybridoma facilities has greatly furthered production of

monoclonal antibodies derived from proteins and peptides generated by the procedures described above.

Protocols for incubating the blotted proteins on nitrocellulose with the primary antibody (immunoblot) vary widely ( 15). Options for detection via a secondary antibody

include colorimetric detection, binding of 125I-labeled protein A, or the very sensitive method in which the secondary antibody provides a luminescent signal detected

by x-ray film (or a luminescence detector). The latter approach has become very widely used. In Figure 36.3, GLUT4 (see Chapter 3) protein levels in transgenic mice

are determined by 125I-labeled protein A which binds to anti-GLUT4 antibody ( 18). The response to high-fat or high-carbohydrate diets shows that overexpression of

the GLUT4 transgene results in considerably more transporter protein being produced.



Figure 36.3. SDS polyacrylamide gel electrophoresis of proteins from muscle (gastrocnemius) or adipose (white adipose tissue) homogenates from transgenic mice

that overexpress the GLUT4 gene. Western blotting: proteins were transferred to membranes, incubated with antiserum against GLUT4 protein, and detected with

125I-protein A. The transgenic mice were fed control (–), high fat or high carbohydrate (+) diets for 14 weeks. (From Ikemoto S, Thompson KS, Takahashi M, et al.

Proc Natl Acad Sci USA 1995;92:3096–9, with permission.)



Immunohistochemistry and Immunocytochemistry

Advances in microscopy have dramatically increased the use of fluorescent antibodies for intracellular localization of specific proteins. Epifluorescence, confocal, and

3D deconvolution microscopic techniques can all take advantage of fluorescently tagged antibodies. Flow cytometry can also be used effectively in nutrition-related

research, particularly that focused on immunology ( 19). In the future, these approaches will see further application in experiments focused on nutrition, especially in

human studies in which sample sizes are small.



Northern Analysis

The universal availability of cloned gene sequences through computer-searchable databases provides the opportunity to use this information to identify nutritionally

regulated genes (13). Northern analysis (northern blotting) gives researchers interested in examining genes that are nutritionally regulated a powerful experimental

technique. Using DNA sequence information, it is possible to produce oligonucleotides (oligomers, oligos) that can be used directly for northern analysis or, as

described below, as primers for production of complementary DNA (cDNA) probes by polymerase chain reaction (PCR) methods. Alternatively, there are commercially

available probes for specific genes, and most investigators are willing to share cDNAs for use as probes for northern analysis studies.

The task of identifying which genes are regulated by nutrient intake level and specific diet formulations or consumption patterns is formidable indeed. Northern

analysis has clearly been a workhorse for these studies to date. Hundreds of examples could be cited. The original method has been modified innumerable times ( 15).

Similarly, tissue RNA extraction procedures have evolved, and currently, guanidinium isothiocyanate–based methods, some with proprietary formulations, are almost

universally used.

The purpose of northern analysis is to detect a specific mRNA and determine its relative static abundance. The method involves agarose gel electrophoresis (AGE) of

the RNA extract, transfer to a filter (nylon or nitrocellulose), binding the RNA to the filter by UV-induced cross-linking, and hybridization to a 32P-labeled DNA probe

(15). The hybridization is detected by autoradiography or phosphorimaging instrumentation, but nonradioactive alternatives are evolving. Intensity of the RNA-DNA

hybridization is usually quantified by video densitometry. Another constitutively produced mRNA should be concurrently examined as a control for equal loading of the

RNA extracts being compared. b-Actin and glyceraldehyde-3-phosphate dehydrogenase cDNAs are frequently used for comparative purposes. Ethidium bromide

staining of RNA separated by AGE prior to transfer can also be used. Results of typical northern blot analyses are shown in Figure 36.4. In panel A, leptin mRNA

levels (see Chapter 87) are lower in fasted rats than in those refed and/or given insulin ( 20). In panel B, metallothionein mRNA levels (see Chapter 11) are virtually

undetectable in zinc-deficient rats compared with levels in rats fed adequate amounts of zinc ( 21). Here, northern blot data are used to assess nutritional status.



Figure 36.4. A. Northern analysis of total RNA from adipose tissue of rats showing the effects of food intake and insulin on leptin mRNA ( ob gene) expression. b-Actin

mRNA is the control for gel-loading efficiency. Left panel shows the relative abundance of the leptin mRNA as determined by densitometry as well as the plasma

glucose concentration. Right panel shows the actual northern blot of leptin and b-actin mRNA. (From Saladin R, De Vos P, Guerre-Millo M, et al. Nature

1995;377:527–9, with permission.) B. Northern analyses of total RNA from rats showing the effect of zinc deficiency (–Zinc) on kidney metallothionein mRNA,

compared with rats fed adequate amounts of zinc ad libitum (Control) or pair fed. b-Actin is the control mRNA. (From Blanchard RK, Cousins RJ. Proc Natl Acad Sci

USA 1996;93:6863–8, with permission.)



As described in the section on antisense techniques, solution hybridization and RNase protection assays provide an alternative to northern analysis for estimating the

mRNA abundance of a gene suspected of being influenced by nutrition. Dot blots are occasionally used in screening experiments; however, since no size separation

of the RNA is involved, the method requires that the probe used produces a signal specific for the mRNA of interest ( 15). Recent advances in the technique of in situ

hybridization using fluorescent probes allow estimation of the intracellular abundance of a specific mRNA ( 22).

Polymerase Chain Reaction

The PCR technique has been used in experiments focused on nutrition and gene expression. PCR can provide multiple copies of a DNA or RNA sequence. Two

oligonucleotide primers (a 5' primer and a 3' primer) that span the targeted sequence of interest must be synthesized ( 15, 23). Repeated sequential polymerase

reactions of the target sequence in both directions under controlled temperatures with a thermocycler allow the targeted sequence to be amplified as a

double-stranded cDNA copy in sufficient quantity for further use. Frequently, the amplified DNA is cloned by standard methods and purified by AGE as needed ( 15,

24).

Reverse transcriptase–PCR (RT-PCR) is used to produce cDNA copies of mRNA. The mRNA is first converted to cDNA with reverse transcriptase, using a specific 3'

primer for the target mRNA (25). PCR is then performed with another specific primer to provide sufficient quantities of the DNA sequence of interest for purification

and/or analysis by AGE. RT-PCR is usually semiquantitative at best, since the amplification process is exponential and differences in mRNA levels usually

encountered with nutritionally regulated genes are not large enough for the technique to be widely applicable. Nevertheless, RT-PCR is valuable for production of

cDNAs for use in northern analysis experiments ( 26). Availability of cDNA sequence information allows development of primers for production of a specific cDNA by

PCR and subsequent cloning to retain the cDNA for labeling and use in hybridizations as needed.

In contrast, competitive RT-PCR (C-RT-PCR) provides a measure of amplification efficiency and an internal control of known concentration so that the C-RT-PCR

technique is quantitative ( 25, 27, 28). Consequently, this approach is very attractive for nutrition experimentation, particularly in human studies or others in which the

number of available cells is very small ( 29). The steps in C-RT-PCR are shown in Figure 36.5 (30). C-RT-PCR requires construction of a third primer to generate a

cDNA to act as the internal control competitor. In practice, prior to PCR, the mRNA is reverse transcribed soon after RNA isolation, since cDNA is more stable during

storage. The method has been applied to zinc status assessment in humans ( 30).



Figure 36.5. Competitive reverse transcriptase-polymerase chain reaction (C-RT-PCR). Extracted RNA is reverse transcribed to cDNA and, together with a competitor

cDNA of known concentration, is amplified by PCR. Comparison of amplification to the competitor concentration allows determination of the relative abundance of the

original mRNA. (Modified from Sullivan VK, Cousins RJ. J Nutr 1997;127:694–8, with permission.)



PCR can be used to identify allelic differences in genotypes that may respond differently to nutrients. A notable example is the polymorphism in the human vitamin D



receptor (VDR) gene and its corresponding link to bone mineral density (BMD) ( 31, 32). Expression of the osteocalcin gene is regulated by the VDR, a trans-acting

transcription factor that requires 1,25(OH) 2 vitamin D3 (calcitriol) as a ligand for binding to the vitamin D response element of the osteocalcin promoter. For these

experiments, PCR was used to amplify genomic DNA from each subject. Subsequent AGE separation of the DNA, after restriction enzyme digestion, produced bands

of different lengths for the two alleles of the VDR gene. The genotypes were differentiated in this way. This approach was also used to examine the positive effects of

dietary fat modulation of plasma low-density-lipoprotein (LDL) cholesterol in individuals with the apoE4 allele (33). The approach has wide application for genes that

produce differences in the use or actions of nutrients and thus serve as biomarkers for differences in nutritional effects.

Promoter Analysis

A number of experiments within the past decade have shown direct interactions between specific nutrients and transcription factors regulating the expression of

specific genes. Metabolites of fat-soluble vitamins, trace metals, sterols including cholesterol, and fatty acids are examples. In each case, an initial interaction

between the nutrient and a receptor or transcription factor(s) is followed by a direct interaction of the transcription factor with specific nucleotide sequences within the

nucleus. Typically, this occurs in the promoter/regulatory region located upstream from the start site of the coding sequence.

The approach taken to examine transcriptional regulation generally follows one of two techniques. The first is the nuclear run-on experiment. This requires generating

purified nuclei from cells or tissues from animals fed a specific diet or provided a nutrient of interest. The nuclei are incubated in the presence of a- 32P-UTP to produce

a radioactive RNA transcript. The transcript is then detected by hybridization to a specific probe ( 15). Since this 32P-nucleotide is only incorporated into mRNA during

transcription, the change in the rate of transcription produced in response to the nutrient is believed to reflect regulation at the level of transcription. A number of

genes for hepatic proteins involved in carbohydrate and lipid metabolism have been shown by nuclear run-on assays to have altered transcription rates in response to

a high carbohydrate diet (34, 35). However, these experiments provide little information about mechanisms involved in transcriptional regulation.

Another approach to promoter analysis enables determination of specific sequences involved in gene expression but requires sequence information about the genes

of interest. In this technique, the promoter region, usually a few thousand bases (kb) of the 5' flanking region, is excised with appropriate restriction enzymes and

ligated to a reporter gene. Usually the chloramphenicol acyltransferase (CAT) or b-galactosidase (b-Gal) genes are used as reporters. The heterologous

promoter-reporter construct must be transfected into a cell type of interest. The cell type is critical since transcription factors are specific to certain cells. Reporter

genes generate products that may be analyzed by liquid scintillation counting or thin layer chromatography (for CAT) or colorimetrically (for b-Gal). The approach has

been used for a number of nutritionally relevant genes ( 11A, 11B, 34, 36, 37 and 38). Promoter/reporter gene systems can be used as an in vitro model for examining

the responses of a promoter to specific nutrients, hormones, or drugs. The leptin gene promoter/reporter construct is an example of this potential ( 39).

Using deletion analysis with restriction enzymes, the location of the nucleotide sequence(s) of a promoter that is essential for the nutrient regulation can be identified.

Mutational analysis of the promoter can define the exact nucleotides needed for specific regulation. Frequently, the latter analysis shows that the response element

(sequence) is not always completely uniform. Consequently, response elements are usually reported as consensus sequences.

Differential Hybridization

The technique of differential hybridization, or plus-minus screening, allows comparison of a gene's expression with two dietary treatments or different periods of

development (40, 41). A plasmid cDNA library is produced by reverse transcription of mRNA from one of the treatment groups. The cDNA is made double-stranded,

ligated into a vector, and used to transform competent E. coli. In addition, a single-stranded cDNA probe is separately generated from mRNA of the other treatment

groups. After E. coli-containing recombinant plasmids are grown with antibiotic selection, the plasmid DNA is cross-linked to nylon filters. The DNA-containing filters

are hybridized separately with the single-stranded cDNA probes (after 32P labeling). The signals generated in autoradiographs will be similar except in a few cases.

The latter potentially represent differentially expressed genes. The colony hybridization procedure is usually repeated for confirmation that a signal is different with the

two different probes. Finally, a northern analysis is carried out using cDNAs from the bacterial colonies in which differential signals originated. These cDNAs must

then be sequenced and searches of genome databases (e.g., the BLAST search program for GenBank) carried out to identify what genes the recovered sequence

represents. This procedure has been used for intestinal genes responsive to dietary zinc intake ( 41).

Differential mRNA Display

An elegant approach to examine differential gene expression was developed in 1992. Differential mRNA display is based on reverse transcription combined with PCR.

It enables identification of mRNAs that increase or decrease in quantity in response to various conditions ( 42). Differential display was designed initially to identify

genes differentially expressed in transformed and nontransformed cells. Recently, the technique has been used to identify genes regulated or influenced by

micronutrients: zinc (21, 43, 44), selenium (45), and copper (46, 47).

Isolation of RNA from tissues is the first step in this technique. Reverse transcription of the RNA uses an oligo d(T) primer that has two non-T bases at the 3' end. This

is the “anchored primer” because it anchors cDNA synthesis at the end of the poly(A) tail and 3' untranslated region. Four anchored primers, each with a different

nucleotide, are used independently for cDNA synthesis to obtain the full array of sequences that can be displayed ( Fig. 36.6). The anchored primer used for the RT

reaction and one of 24 decanucleotide primers (arbitrary primers) are used for cDNA amplification by PCR. These constitute the 3' and 5' primers, respectively. A 32Por 33S-labeled nucleotide is included in this PCR reaction. The resulting labeled cDNA then represents one of 24 possible subsets of mRNAs from the original RNA

used for analysis. The four anchored primers, when used with the 24 arbitrary primers, require 96 separate PCR reactions to provide a thorough differential display

from a given tissue or cell type. A few mRNAs may not be recognized because of a lack of sufficient poly(A) tail sequence or poor priming with the random primers

used. As shown in Figure 36.6, differential mRNA display can be viewed as a library with four floors (one for each anchored primer) and 24 shelves with many books

(one for each arbitrary primer). When all shelves on all floors have been read, all of the information has been viewed.



Figure 36.6. Differential mRNA display. Cell RNA is converted to cDNA, which is amplified by PCR using one anchored primer (3') and one arbitrary primer (5').

These are analogous to a floor and stack in a library, respectively. The cDNA products are then separated by polyacrylamide gel electrophoresis and compared as

shown in Figure 36.7.



Figure 36.7. Differential display of intestinal mRNA from zinc-deficient rats. The mRNA is converted to cDNA by reverse transcription. cDNA is amplified by PCR with

one of four anchored primers (3') and one of 24 arbitrary primers (5'), followed by PAGE of the cDNAs run in duplicate (as outlined in Fig. 36.6). RNA was from rats

fed a zinc-deficient (–Zn) diet or zinc-adequate diet provided ad libitum (+ Zn) or pair fed (P.F.). The display series to the left shows a cDNA (A) derived from an mRNA

increased in –Zn while the display series to the right shows a cDNA (B) derived from an mRNA decreased in –Zn. (From Blanchard RK, Cousins RJ. Proc Natl Acad

Sci USA 1996;93:6863–8, with permission.)



Denaturing polyacrylamide gel electrophoresis (PAGE) and autoradiography allow separation and isolation of individual cDNA bands ( Fig. 36.7). Bands are usually up

to 400 bp, but newer variations of the original method may significantly increase that size, thus increasing the probability of sequence identification. The cDNA bands

resulting from mRNAs of animals fed under differing dietary conditions are resolved by PAGE and those that differ in intensity are considered manifestations of altered

levels of a specific mRNA (i.e., one that is differentially expressed). In the example, there are cDNAs that increase and decrease in relative abundance during zinc

deficiency (21).

Subsequently, the cDNA band from a differentially expressed mRNA is cloned into a plasmid to maintain a renewable source of the cDNA. Verification of differential

expression requires northern analysis using the cDNA in question (after 32P labeling) and RNA from other experimental animals subjected to the same dietary

conditions. If northern analysis confirms differential expression, this cDNA is sequenced, and its identity is determined through genome database searches as

described above. Some searches do not reveal any previously reported sequences. Other searches identify a specific gene sequence, a gene of close homology, or

an expressed sequence tag (EST). The latter represents a partial cDNA sequence of an unidentified gene. As more of the genomes of humans and experimental

animals are sequenced, the EST data will become more useful.

Positional Cloning

An excellent example of positional cloning is that of the ob gene (48). This led to identification of a new peptide hormone, leptin, which, when secreted by adipocytes,

regulates food intake and energy expenditure and influences non-insulin-dependent diabetes in obese individuals ( 49). This research was based on observations of

mutations in mouse strains made over four decades earlier. (See Chapter 87 for more information.)

The first step in this approach is obtaining genetic maps to identify the locus of the mutation of interest. In many cases the chromosomal location of a mutation is

known in the murine and human genomes. For genes that influence nutrition, phenotypic expression (i.e., the development of obesity) is used to segregate the mutant

gene through specific genetic crosses. Linkage studies position the gene relative to molecular markers or known/identifiable areas of the genome. Establishing

linkages to known genes helps greatly, particularly if they are “tightly linked” to the gene of interest. In the case of the ob gene, the region of chromosome 6 where the

ob gene was located had been identified by two flanking markers. Using this location information, the appropriate region of the DNA is cloned. The use of yeast

artificial chromosomes (YACs), specific restriction enzyme digests, and specific crosses refine the location. Subsequent screening of restriction digests will yield

smaller sections of DNA, some of which will hybridize to RNA from a tissue in which the gene is expressed. Using this DNA as a probe, the mRNA of interest is

identified, the cDNA sequence of the gene can then be established, and eventually, as in the case of leptin, the primary sequence of protein product deduced ( 48).

Experiments of this type are extremely time consuming and expertise in molecular genetics is essential. Nevertheless, the results have great benefit for understanding

important aspects of nutrition. The fruits of such efforts are usually extensive, rapid, and enlightening. A database search for leptin reveals recent studies on the use

of leptin protein and analytical assays ( 50), production of corollary animal models ( 51), therapeutic opportunities ( 49, 52), and additional components, such as

neuropeptide Y (53), of the system of body composition regulation.



MANIPULATING GENES REGULATED BY INDIVIDUAL NUTRIENTS OR DIETARY PATTERNS

Transgenic Animals

The opportunity to overexpress a gene and thus increase production of a gene product has significant potential for nutrition research. Genes responsive to specific

nutrients can be beneficial or deleterious when overexpressed.

The transgenic overexpression technique involves production of a construct consisting of a promoter and structural gene. The promoter can be the gene's normal

promoter (homologous) or a different promoter (heterologous). A purified sample of the construct is injected into fertilized eggs (usually murine or porcine) and, if the

construct DNA becomes integrated into the genome of some eggs, transgenic animals will be produced from them after full gestation in foster mothers. Southern

(DNA) blotting or PCR is required for detection of the unique DNA construct to initially distinguish transgenic animals from their nontransgenic littermates. Selective

breeding can produce homozygous lines of animals carrying transgenes.

A salient example of the transgenic approach used the metallothionein gene promoter to direct expression of the growth hormone (GH) gene in mice and pigs ( 54, 55).

In pigs, additional GH was produced and secreted that greatly increased lean body mass. Since the metallothionein gene is induced by dietary zinc ( 56), adding extra

zinc to the food or drinking water was used to increase expression of the transgene. This approach was used to produce transgenic animals with a construct

composed of the metallothionein promoter and LDL receptor structural gene ( 57). Injection of the inducer metal activates the promoter and yields a lowered circulating

cholesterol level within hours, coincident with increased production of the LDL receptor protein.

Other examples of the use of transgenic animals to address questions of nutritional interest are available. The glucose transporter (GLUT4) was overexpressed in

mice using the aP2 fatty acid–binding protein promoter and a genomic DNA fragment containing the entire human GLUT4 gene as the construct ( 58). Overexpression

was detected by increased GLUT4 protein in membranes from both white and brown fat ( Fig. 36.8). These transgenic mice also had higher glucose transport rates,

lower glucose tolerance curves, and more body fat than littermates. In contrast, overexpression of metallothionein, a zinc/copper-binding protein, did not produce very

dramatic gross effects on body composition or growth (59) but influenced zinc homeostasis ( 60). The genes for phosphoenolpyruvate carboxykinase, copper/zinc

superoxide dismutase, hepatic lipase, lipoprotein lipase, and several apolipoproteins (see Chapter 74 and Chapter 75) have been overexpressed in transgenic mice

(61, 62, 63, 64, 65, 66 and 67). Table 36.1 lists some genes of nutritional interest that have been overexpressed in transgenic mice.



Figure 36.8. Overexpression of the human GLUT4 gene in transgenic mice. Top panel, the transgene construct consisting of the mouse aP2 fatty acid-binding

promoter ligated to 6.3 kb of human genomic DNA that includes all 11 exons of the glut4 transporter gene. Bottom panels, Growth and body composition comparisons

for control (crosshatched bars) and GLUT4 transgenic mice (solid bars) fed a normal diet. (From Shepherd PR, Gnudi L, Tozzo E, et al. J Biol Chem

1993;268:22243–6, with permission.)



Table 36.1 Transgenic Mice Overexpressing Genes of Interest in Nutrition Research—Examples



An encouraging aspect of the application of transgenic animals for nutrition research is that as these strains become more available through the Jackson Laboratory

and other specialized distributors or from individual investigators, major new insights into nutrient metabolism and function will emerge.

Gene Knockout (Null Mutation) Animals

Gene knockout technology provides the opportunity to delete expression of a specific gene (null mutation). As a result, the normal gene product is not produced.

Consequently, this technology is the engineered counterpart to spontaneous mutations that occur in laboratory animals and are propagated by selective breeding.

The ob gene mutation of mice is an example of a spontaneous mutation.

Knockout technology in animals has, thus far, been applied exclusively to murine genes. In some cases, the animals die prior to birth if a critical gene product is not

produced. Numerous examples of this phenomenon have been reported. Deletion of the C/EBPa gene, which is required for energy (glucose) utilization, is an

example relevant to nutrition (68). In some instances of deletion of a gene that may appear critical for nutrient metabolism (e.g., metallothionein for zinc metabolism),

mice bearing the null mutation still reproduce and develop normally, albeit metabolic abnormalities can be demonstrated ( 60, 69). Similarly, a null mutation in the

cellular retinoic acid–binding protein I gene (CRABPI), which is believed to be required for the function of vitamin A (retinol) via the metabolite retinoic acid, produces

animals that appear to have a normal phenotype (71). These latter results indicate that, in some instances, another gene product provides the same phenotypic effect

through redundancy of functional roles of specific genes or a gene family. Redundancy of function can obscure the true function of the deleted gene. Deletions of the

genes for b2-microglobulin, metallothionein, a-lactalbumin, Cu/Zn superoxide dismutase, cellular glutathione peroxidase, and apolipoprotein E have been reported to

have metabolic consequences (72, 73, 74, 75, 77, 78 and 78a). Table 36.2 shows examples of nutrient-related consequences detected in mice with null mutations.



Table 36.2 Knockout (Null Mutation) Mice with Deleted Genes of Interest in Nutrition Research—Examples



In an interesting extension of the knockout technique, transgenic mice overexpressing apolipoprotein A-I were bred to apolipoprotein E null mice. The offspring had

higher HDL levels and increased atherosclerotic lesions ( 79). Similarly, a mouse model with features of familial combined hyperlipidemia can be produced by crossing

transgenic mice carrying the human apolipoprotein C-III to mice null for the LDL receptor ( 66).

The technique of creating a knockout animal model or null mutation is more correctly called “gene targeting by homologous recombination.” Animal cells are diploid,

i.e., they contain two copies (alleles) of a gene. The targeted gene is disrupted in one allele (producing heterozygotes with the null mutation), and homozygous

genotypes are then produced by selective breeding of the heterozygous strain.

There are two approaches to developing knockout mice. The original approach is to isolate the murine gene under investigation, identify the exons by mapping, delete

part of an exon, and replace it with the gene encoding neomycin resistance (thus producing a marker for selection). An entire exon can also be deleted. This construct

is the gene targeting vector. The targeting vector is linearized and transfected into embryonic stem (ES) cells by microinjection or electroporation. The transfected

cells are then injected into the blastocysts of mice at 3.5 days of pregnancy and introduced into pseudopregnant mice. Chimeric pups are often identified by the agouti

coat color provided by genes from the ES cells. Selective breeding to obtain a null mutant (– / –) follows ( 15, 83).

The second and more recent approach allows cell type–specific targeting of a gene deletion ( 84, 85). The gene is engineered to have specific sequences (lox P sites)

on either side and, by ES cell technology, a transgenic line is created carrying the target gene and flanking lox P sites. Another transgenic strain is produced with a

construct comprising the CRe recombinase gene and a promoter (e.g., Mx1) that can be activated by an inducer (e.g., Interferon-a [IFNa]) in a tissue-specific manner.

These mouse strains are crossed and, when IFNa is injected into offspring, the CRe gene product (CRe recombinase; a bacteriophage enzyme) acts at one of the lox

P sites, deleting the target gene in a specific tissue/cell type where the CRe gene product is produced ( 84, 85).

These techniques, while specialized, are yielding murine strains that will be generally available for nutritional and metabolic studies. As with transgenic

overexpressing mice, commercial suppliers may act as a resource for such animals.

Inhibition of Specific Gene Expression by Antisense Oligonucleotides and Transgenes

Antisense RNA has recently been used as a research tool for questions of nutritional interest. There are three ways in which antisense sequences can be used.

Computer searches of the literature usually do not differentiate between these uses.

The first is the use of antisense RNA as probes in solution hybridization/RNase protection assays. When sequence information is known, specific oligonucleotides

can be generated and used to obtain information about the relative abundance of a specific mRNA. Tissue RNA is hybridized with the 32P-labeled antisense RNA

probe, treated with RNase to hydrolyze single-stranded RNA, and then the protected 32P-labeled RNA (hybridized RNA) is precipitated and separated by

electrophoresis followed by autoradiography. Abundance is measured by densitometry. Once the assay has been established, the precipitated 32P-labeled hybrid is

measured by liquid scintillation counting for comparison of mRNA abundance. This approach has been used to examine GLUT4 transporter expression and glucagon

receptor expression (86, 87).

The second application of antisense oligonucleotides is selective permanent inhibition of a targeted RNA ( 88). Usually, oligonucleotides are at least 12 to 25

nucleotides long to provide the specificity needed for identification of a unique mRNA. Sites where targeting is most effective are the double-stranded regions of

secondary mRNA structure. This approach has been used in experiments with cells. To address directly questions of nutritional interest, antisense sequences can be



used to generate transgenic mice. The antisense sequences are introduced by the construct used for microinjection. Mice carrying the transgene continually produce

antisense sequences that neutralize the effect of the targeted mRNA, thus preventing formation of the gene product. Consequently, these transgenics have altered

phenotypes. This approach has received limited use in experiments focused on nutrition and gene expression ( 89, 90).

The third use of antisense sequence information is for transient inhibition of translation of specific mRNAs by hybridization. This approach has received attention

because of the potential therapeutic use of antisense oligonucleotides. The exact mechanisms of their action are also actively investigated (reviewed in [ 88, 91]).

These oligonucleotides appear to be taken up by cells in the brain and some tissues. The approach has been recently applied to inhibition of mRNAs for peptides

involved in regulation of feeding behavior and body weight ( 92, 93, 94 and 95). In these cases, antisense DNA sequences are introduced into specific areas of the

brain. Examples of use of this technique are shown in Table 36.3.



Table 36.3 Antisense Oligonucleotide-Inhibited Gene Expression—Examples



SUMMARY

The area of nutrition and gene expression is gaining interest rapidly and is now a recognized research discipline in nutritional sciences. As our knowledge of animal

and human genomes expands, the technologies described here and new approaches still to be developed will have a profound impact on nutrition as a field and on

our understanding of how the diet influences genetic expression.



ACKNOWLEDGMENTS

The author expresses his appreciation to Drs. Barbara A. Davis of Virginia Polytechnic Institute and State University and Cathy W. Levenson of Florida State

University for their review of this chapter and valuable comments, and to Mr. Walter M. Jones for drawing some of the figures.

Abbreviations: PCR—polymerase chain reaction; PAGE—polyacrylamide gel electrophoresis; RT—reverse transcriptase; AGE—agarose gel electrophoresis.



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Chapter 37. Transmembrane Signaling: A Tutorial

Modern Nutrition in Health and Disease



Chapter 37. Transmembrane Signaling: A Tutorial

ROBERT A. GABBAY and JEFFREY S. FLIER

Receptors

Receptor Types

G-Proteins Linked to Ion Channels Directly

Receptors as Enzymes

Transmembrane Signaling Defects Leading to Disease

Chapter References

Selected Readings



The evolution of life forms from unicellular to multicellular organisms necessitated a mechanism for cells to communicate with each other. One of the primary modes of

cellular communication is the release of soluble factors from one cell or organ to influence the metabolism and biology of another. These soluble factors—which

include hormones, neurotransmitters, growth factors, and cytokines—are generally hydrophilic molecules that cannot cross the hydrophobic plasma membrane of

cells. A system is therefore required to recognize these extracellular signals and transmit the information across the plasma membrane to direct appropriate changes

in the intracellular environment. This tutorial focuses on the basic principles underlying this signaling system, with an emphasis on those mechanisms pertinent to

physiology and nutrition. Then, we see how aberrations in this signaling system can result in diabetes, cancer, and a host of other metabolic disturbances.

Any transmembrane signaling system requires several features. First, there must be specificity in responding to a given extracellular signal. Recognition of this signal

will then generate a specific response. This response should also be specific to the cell type or organ involved. Next, the response to a specific signal must be rapid.

An efficient mechanism is required to shut off that signal when it is no longer needed. Finally, there must be a means of regulating a host of processes that will

ultimately produce the biologic response desired ( 1).

Extracellular signals are recognized through their specific binding to cell surface receptors. These receptors, generally transmembrane proteins, interact with an

intracellular effector system that often results in production of small soluble molecules (second messengers) that diffuse through the cell to mediate the actions of the

given extracellular stimulus ( Fig. 37.1). The concept of a second messenger was first proposed by Sutherland in 1959 when he identified cyclic AMP as a second

messenger capable of mimicking the effects of the hormone epinephrine. Sutherland proposed that the hormone is the first messenger released into the bloodstream

to interact with a specific membrane receptor on the target tissue. The specific binding of the hormone to its cell-surface receptor leads to generation of a second,

intracellular messenger that then mediates the intracellular actions of the given hormone. This model of hormone action has been borne out for many different

extracellular signals. Although there are few second messenger systems, they are used by a myriad of different extracellular stimuli. Along with cyclic AMP, the other

main second messengers mediating hormone action include cyclic GMP, calcium, and diacylglycerol. In most cases, GTP-binding proteins (G-proteins) act as

molecular switches to couple receptor binding to the enzymes that generate the specific second messenger. All these signaling pathways share a final common

mechanism in activating protein kinases that alter subsequent enzyme activities through covalent addition of a phosphate group to serine, threonine, or tyrosine

residues of various proteins. Although many hormones act through the generation of second messengers that then stimulate specific protein kinases, some directly

lead to protein kinase activation by binding to receptors that have intrinsic tyrosine kinase activity. Regardless of the route leading to protein kinase activation,

subsequent changes in protein phosphorylation alters the activity of proteins involved in nutrient flux, transcription of specific genes, protein synthesis, and cellular

trafficking of proteins.



Figure 37.1. General schematic overview of ligand-receptor pathway of biologic effects.



The steroid hormones are more hydrophobic and capable of crossing the plasma membrane. They bind specifically to a soluble receptor in the cytoplasm. The

hormone-receptor complex then enters the nucleus to function as a DNA-binding protein that directly affects transcription of specific genes. We will not cover this

nuclear hormone signaling mechanism here but, instead, will focus on transmembrane signaling at the plasma membrane. The prototypical signaling pathways

through which the vast majority of other hormones act and, finally, how aberrations in these signaling mechanisms may underlie diseases from cancer to diabetes are

described in detail below.

The rapid advances in understanding transmembrane signal transduction have been aided by the use of several powerful techniques. Recombinant DNA technology

(see Chapter 36) has permitted the rapid cloning of members of signaling families, i.e., structurally and functionally related groups of proteins. The hundreds of

different receptors can be grouped into only a few large families of receptor types. The introduction of several mutants and inhibitors of signaling pathways into

cultured cells has allowed further dissection and understanding of the transmembrane signaling pathway. Finally, the development of transgenic animals in which a

particular gene and protein can either be overexpressed or “knocked-out” allows integration of cellular signaling pathways with physiologic studies.



RECEPTORS

The initial step in transmembrane signaling involves the binding of the extracellular signal or hormone to a cell-surface receptor. Receptors provide the first level of

specificity by their presence on only certain tissues and cell types. These receptors have several characteristics that poise them for recognition of extracellular signals

(2). First, they have high affinity for the ligand (hormone or other extracellular signal that binds to the receptor). This is necessary because most hormones circulate in

the bloodstream at concentrations below 10 –8 M. This high affinity enables cells to respond to small changes in hormone concentration. Receptors are also specific for

their ligand or hormone. This means that insulin binds only to the insulin receptor, whereas glucagon binds only to the glucagon receptor. Various structural

analogues of a given hormone bind to their receptor with differing affinities that correspond to their biologic activity. For example, insulin binds to the insulin receptor

with 50 times higher affinity than proinsulin and is 50-fold more potent than proinsulin in stimulating glucose uptake. Receptor binding can be plotted as shown in

Figure 37.2. Agents can be found that have higher affinity for a receptor (potent agonists), whereas others may bind to receptors with high affinity but not trigger the

usual transmembrane signaling and biologic responses (antagonists). These principles have led to the development of a large number of widely used pharmacologic

therapies.



Figure 37.2. Concentration dependence of the effects of an agonist and an antagonist relative to native hormone on a typical biologic response.



Hormone-receptor interactions can be studied by a variety of techniques. The receptor number and affinity on a particular target cell is classically measured using a

Scatchard analysis (Fig. 37.3). By allowing labeled hormone to compete with unlabeled (cold) hormone for receptor binding, the amount of hormone bound to receptor

is determined at a number of hormone concentrations. This amount of bound hormone is plotted against the ratio of bound to free hormone, typically yielding a

straight line. The slope of this line is the negative value of the association constant ( –Ka), which indicates the receptor affinity for the hormone or ligand. The intercept

on the abscissa represents the total receptor concentration (R T).



Figure 37.3. Diagram of Scatchard analysis, showing the relationship of slope to –K a and of the intercept to the total number of receptors.



A given hormone can bind to several different receptors, each of which communicates with a unique intracellular effector system. For example, epinephrine can bind

to both a- and b-adrenergic receptors. b-Adrenergic receptors are coupled to a cyclic AMP–generating second-messenger system, whereas a-adrenergic receptors

use calcium as their second messenger or act by decreasing the level of cyclic AMP ( 3). Further specificity of action is obtained by the presence of various receptor

subtypes located in different tissues. For example, b 1-adrenergic receptors are present primarily in the heart, where they function to stimulate heart rate and increase

the force of cardiac contraction. b 2-Receptors, on the other hand, are primarily located in the lungs, where they cause bronchial dilation. This specificity in receptor

subtype tissue distribution has been used to develop specific pharmacologic agents with high affinity for particular receptor subtypes (e.g., b 1-adrenergic

receptor–blocking agents that selectively decrease heart rate without causing bronchoconstriction or b 2-agonists that cause bronchial dilation with minimal effects on

cardiac function).

The ligands that bind to receptors can take many forms. Initially, the study of receptors focused on protein hormones as the ligands of interest. Since that time, a

variety of ligands including neurotransmitters, cytokines (proliferating and differentiating agents of the hematopoietic and immune systems), and various growth

factors also have been shown to act through receptor binding to specific cell-surface receptors. Metabolites also can act as ligands through binding to specific

receptors. For example, fatty acids released by fat metabolism can bind to a specific transcription factor receptor (peroxisome proliferator-activating receptor, PPARg)

to stimulate specifically adipose cell differentiation. Another “ligandlike” agent is light, which, through photoisomerization of 11- cis-retinal, stimulates a specific

receptor in the eye to begin the transmembrane signaling cascade responsible for vision (see Chapter 17).

Receptor Types

Receptors can be divided into four major types: (a) G-protein-linked receptors; (b) enzyme-linked receptors; (c) ion-channel-linked receptors; and (d) steroid hormone

receptors. G-protein-linked receptors use a GTP-binding protein to couple the receptor to the particular effector system that, typically, generates a second messenger

(i.e., cyclic AMP, cyclic GMP, inositol trisphosphate, or diacylglycerol). Enzyme-linked receptors either have intrinsic enzyme activity or are tightly coupled to proteins

with enzyme activity. As discussed above (see Chapter 17 and Chapter 18), steroid/retinoid hormone receptors are located in the cytoplasm and/or nucleus where

they recognize steroid hormones that have diffused across the plasma membrane. These receptors still possess the same basic properties of receptors described

above (i.e., high affinity and specificity), but they have several unique features as described in other chapters. Ion channel receptors, also termed ligand-gated

channels, act to increase ion flux (usually calcium, potassium, or sodium) across the plasma membrane, either as direct conduits for ion flow or through coupling to

ion channels (see Chapter 38). The following sections focus on the G-protein-linked and enzyme-linked receptors.

G-Proteins

Over the last 25 years, many receptors linked to effector systems by trimeric GTP-binding proteins (G-proteins) have been described. These proteins regulate the

activity of a specific plasma membrane enzyme or ion channel in response to receptor binding. This transmembrane signaling pathway, first described by Gilman,

uses a trimeric GTP-binding protein as a molecular switch to activate the appropriate effector system rapidly. Hormone binding leads to G-protein activation through

the binding of GTP. The G-protein, which is active in the GTP-bound state, can then move through the plasma membrane to modulate the appropriate effector system.

G-Proteins have an intrinsic GTPase activity that hydrolyzes the bound GTP to GDP, inactivating the G-protein. This permits both the rapid turning on (binding of

GTP) and turning off (hydrolysis by GTPase) of the signal that is needed to respond to rapid fluctuations in hormone levels.

Several different types of G-proteins couple to specific plasma-membrane enzyme-effector systems leading to generation of soluble second messengers ( 5). Cyclic

AMP was the first second messenger discovered. Subsequent research showed that a specific G-protein ( Gs) couples receptor binding to the activation of the enzyme

that generates cyclic AMP (adenylate cyclase). As a second messenger, the cyclic AMP that is generated mediates all the intracellular actions of the hormone. The

rapid generation of cyclic AMP can be turned off by hydrolysis with phosphodiesterase, providing a transient response to a given signal ( Fig. 37.4). In addition, the G

protein has an intrinsic GTPase activity that hydrolyzes the GTP bound to G s, restoring Gs to the inactive form. Finally, a different G-protein (G i), when activated by

other receptors, leads to direct inhibition of adenylate cyclase activity and, thus, cyclic AMP generation. A host of hormones act through this G s–adenylate

cyclase–cyclic AMP mechanism (Fig. 37.4).