Annals of Medicine

ISSN: 0785-3890 (Print) 1365-2060 (Online) Journal homepage: http://www.tandfonline.com/loi/iann20

Transgenic Animals in Lipoprotein Research Katriina Aalto-Setälä To cite this article: Katriina Aalto-Setälä (1992) Transgenic Animals in Lipoprotein Research, Annals of Medicine, 24:5, 405-409, DOI: 10.3109/07853899209147846 To link to this article: http://dx.doi.org/10.3109/07853899209147846

Published online: 08 Jul 2009.

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Special Section: MoJecular Genetics and Genetic Epidemiology of Cardiovascular Disease and Diabetes

Transgenic Animals in Lipoprotein Research

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Katriina Aalto-Setala

Lipid metabolism is a complex system involving the interaction of several lipid-transporting and -transferring serum proteins, enzymes regulating lipoprotein synthesis and modification, and lipoprotein receptors. Several epidemiological studies have demonstrated that many abnormalities in lipid metabolism are associated with coronary heart disease risk. There are at least 17 different genes that code for proteins directly involved in lipoprotein metabolism. Naturally occurring mutations in many of these genes have been described that affect either the quantity or the quality of these proteins. Such genetic alterations aid in the precise evaluation of the function of these gene products. Transgenic animal technology has made it possible to create genetic alterations and to study the consequences of overexpression of a gene and expression of a mutated protein, and also to study species-specific differences between proteins. Key words: apolipoproteins; cholesteryl ester transfer protein; LDL receptor; lipoprotein metabolism; transgenic mice. (Annals of Medicine 24: 405-409,1992)

Introduction Lipoproteins are complexes of lipids and apolipoproteins. Lipids, i.e. triglycerides and cholesteryl ester, form the core of the particles. This lipid core is surrounded by a surface consisting of amphiphilic phospholipids and apolipoproteins to make the hydrophobic lipid core water-soluble. Apolipoproteins (apo A-I, A-11, A-IV, 6,E, CI, CII, Clll and apo (a)) serve as structural proteins, but they also have other important functions, such as the activation of different enzymes and serving as ligands for receptor binding (Table 1) (1). Four types of lipoproteins can be identified in the plasma (Fig. 1). Chylomicrons and VLDL are triglyceride-rich particles. Chylomicrons are formed in the intestine and they are hydrolysed by lipoprotein lipase (LPL) enzyme on capillary endothelia to be converted to remnant particles that are rapidly removed from the circulation by liver. VLDL particles are formed in the liver and they are converted by LPL to intermediate density lipoproteins (IDL) which are further converted by hepatic lipase (HL) to low density lipoproteins (LDL). LDL is finally removed from the bloodstream by specific LDL receptors in liver and also in peripheral cells. Chylomicron remnants and LDL particles are cholesterol-rich and they are known to be atherogenic. High density lipoFrom the Rockefeller University, New York, U.S.A. Address and reprint requests: Katriina Aalto-Setall, The Rockefeller University, 1230 York Avenue, New York, NY 10021, U.S.A.

proteins (HDL) are also cholesterol-rich particles, but they are known to be antiatherogenic. HDL removes free cholesterol from peripheral cells. This free cholesterol is then esterified in plasma by lecithin cholesteryl acyl transferase (LCAT) enzyme. Cholesteryl ester is further transferred from HDL to VLDL and LDL by the action of cholesteryl ester transfer protein (CETP) to be taken to the liver. The liver is the only organ that can remove cholesterol from the body. The introduction of foreign genes into the germ line has made it possible to create genetic alterations in each of the genes involved in lipid metabolism and to study their effects in vivo. The gene of interest must be first isolated and purified. This gene can be under the control of its own promoter or the expression can be controlled by a heterologous promoter like the inducible mouse metallothionine-I promoter. DNA contract is injected into a pronucleus of a fertilized egg and the eggs are implanted into the oviduct of a pseudopregnant foster mother. The incorporation of the transgene is usually analysed from DNA in the tail tips of the offspring. The protein product of the transgene can also be used to identify the positive offspring.

Transgenic Animals in Lipid Research Transgenic animal technique has been used for the last 10 years to study the effects of single genes in vivo (2).

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Table 1. Apolipoprotelns and thelr functions. Apolipoprotein

Particle type

Function

apo A-I

HDL, chylomicrons

LCAT activation Tissue cholesterol efflux

apo A-ll apo A-IV apo B

HDL, chylomicrons HDL, chylomicrons, lipid-free VLDL, LDL, chylomicrons

apo CI apo CII apo Clll apo E

HDL, VLDL, chylomicrons HDL, VLDL, chylomicrons HDL. VLDL, chylomicrons HDL, VLDL, chylomicrons

VLDL and chylomicron production Ligand for LDL receptor Modulate the uptake of TG-rich lipoproteins Lipoprotein lipase activation Modulate the uptake of TG-rich lipoproteins Ligand for LDL and chylomicron remnant receptors

Figure 1. A schematic diagram of lipoprotein metabolism. VLDL, very low density lipoprotein; IDL. intermediate density lipoprotein; LDL, low density lipoprotein; HDL, high density lipoprotein; LPL, lipoprotein lipase; HL, hepatic lipase; CETP, cholesteryl ester transfer protein; LCAT, lecithin cholesteryl acyltransferase. Transgenic mouse lines so far reported with altered lipoprotein phenotype have been presented in Table 2. All of the mice overexpress the gene of interest and the consequences of the overexpression on lipoprotein metabolism have been studied.

LDL Receptor The first transgenic mice to express a human gene involved in lipid metabolism were human LDL receptor transgenic mice (HuLDLRTg) (3).LDL receptor is essential for the removal of cholesterol-rich LDL particles from the bloodstream. Patients who lack or have decreased numbers of normal LDL receptors have familial hypercholesterolaemia with cholesterol accumulations in tendons, eye-lids and arteries, and they develop coronary heart disease at an early age (4). In HuLDLRTg mice, the expression of the human LDL receptor gene was under the control of the mouse metallothionine-I promoter that is not tissue specific. However, most of the human LDL receptor expression was found in liver. The

plasma cholesterol concentration in HuLDLRTg mice was observed to be decreased from the normal 76 mg/dl to 29 mg/dl. When a LDL turnover study was performed with these mice, their LDL was cleared much faster than in control mice. An important observation was also that the increase in plasma cholesterol by high fat diet was significantly less in the HuLDLRTg mice than in controls (5).

Apolipopro iein A -I The second transgenic mouse line expressing a gene involved in lipid metabolism was human apolipoprotein A-l transgenic mice (HuAITg) (6). Apo A-l is known to be the major protein in HDL, and plasma HDL cholesterol concentration has been shown in many human epidemiological studies to be inversely correlated with atherosclerosis susceptibility, i.e. the higher serum HDL cholesterol value, the lower the risk of atherosclerosis. When a human apo A-l gene was introduced into mice, elevation in serum HDL cholesterol was observed from

Transgenic Animals in Lipoprotein Research Table 2. Transgenic mice with altered lipid or lipoprotein phenotype. Transgenic mouse line

Phenotype ~~~

HuAITg' HuClTg HuClllTg RatETg HuLDLRTg HuCETPTg

HDL 1 TG T TGft T G I CHOLl LDL 1 HDL 1

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'HuAITg, human apo A-l transgenic mice; HuCITg, human apo CI transgenic mice; HuCIIITg, human apo Clll transgenic mice; RatETg, rat apo E transgenic mice; HuLDLRTg, human LDL receptor transgenic mice; HuCETPTg, human CETP transgenic mice.

the normal 55 mg/dl to 90 mg/dl. Normally,.mice have only one size of HDL particle, corresponding to human HDL,, but these HuAlTg mice were found to have an increased amount of small HDL particles, HDL, (6). HuAlTg mice were, thus, found to have two differentsized HDL populations instead of the single size population normally present in mice. Mouse endogenous apo A-l was found to be decreased in HuAlTg mice (7, 8). HDL metabolism was also studied in these HuAlTg mice with HDL labelled with 3H-cholesterylester and '251-apo A-I. The removal of labelled apo A-I represents the uptake of whole HDL particles, whereas the removal of labelled cholesteryl ester represents the selective uptake of cholesteryl ester without the removal of the whole HDL particle. In control mice, both particle and selective uptake of HDL were removed (8). However, in HuAlTg mice, only uptake of HDL particles was observed with no detectable selective uptake. The uptake of HDL particles in HuAlTg mice was the same as that in control mice. This finding suggests that the primary sequence of apo A-l determines the selective uptake of HDL cholesteryl esters. In dietary studies with atherogenic diets, HuAlTg mice have been found to be resistant to atherosclerosis (9). HuAlTg mice developed significantly fewer atheroma plaques than their negative litter mates. This is important additional proof of the antiatherogenic effect of HDL.

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also been created, but their serum lipid levels have been found to be normal (11). This could be due to the fact that the HuETg mice so far analysed express only small amounts of apo E that are not sufficient to demonstrate the lipid lowering effect seen in the rat apo E transgenic mice. It is also possible that there are some speciesspecific differences in receptor binding properties of apolipoprotein E.

Apolipoprotein Clll Apolipoprotein Clll (apo CHI) is present in chylomicrons, VLDL and HDL particles. In in vitfo studies it has been shown to inhibit the tissue uptake of triglyceride-rich particles and also to inhibit the action of LPL. In in vivo studies, apo Clll has been found to correlate with serum triglyceride levels, and a DNA polymorphism in apo Clll gene has been found to associate with serum triglyceride levels. Transgenic mice expressing human apo Clll gene were the first animal model with primary hypertriglyceridemia (12); It was easy to distinguish a positive offspring carrying the human apo Clll gene from a negative litter mate just by looking at the plasma: mice expressing the human apo Clll gene were found to have cloudy, even milky plasma. Only a small increase in plasma apo Clll levels increased plasma triglyceride levels 2-3 times above normal values. This was the first definitive observation of an important function of apo Clll in triglyceride metabolism. Elevated triglycerides in these HuClllTg mice were mainly found to be in the VLDL fraction. Further studies with these mice revealed that their VLDL fractional catabolic rate was greatly diminished (13),and this was found to be due not to the inhibition of LPL, but to the inhibition of the tissue uptake of these triglyceride-rich particles. VLDL particles in these HuClllTg mice were found to be poor in apo E, the apolipoprotein that is known to mediate the binding of the triglyceride-rich particles to their receptors, but they were found to be rich in apo Clll and triglycerides. The total number of VLDL particles in circulation was also increased. Triglyceride synthesis was also found to be increased in these mice by about two-fold, without an increase in the production of apo B. From these studies it was concluded that apo Clll modulates the uptake of triglyceride-rich particles, most likely before they are converted to triglyceride-poor, cholesterol-rich remnants.

Apolipoprotein E Apolipoprotein E (apo E) is present in chylomicrons, in their remnants, in VLDL and in HDL. Apo E serves as the ligand for binding to a remnant receptor, the so-called LDL receptor-like protein (LRP), and it is also capable of binding to the LDL receptor. Mice expressing rat apo E were found to have decreased plasma levels of triglycerides and cholesterol (10). In these transgenic animals, chylomicrons and VLDL particles were cleared from the circulation faster than in their negative litter mates and in a dose-dependent manner. These observations demonstrated the importance of apo E in the clearance of triglyceride-rich lipoproteins. The removal of LDL was also enhanced in these rat apo E transgenic mice. Transgenic mice expressing the human apo E gene (HuETg) have

Apolipoprotein CI Apolipoprotein CI (apo Cl) has been shown to modulate the binding of triglyceride-rich particles to LRP and also to LDL receptors. Transgenic mice expressing human apo CI (HuCITg mice) have been created (14), and these mice were found to have mild hypertriglyceridaemia.Apo CI is located in close proximity to the apo E gene on chromosome 19. Transgenic mice were also created that expressed both human apo E and CI genes. An interesting observation with these double transgenic mice was that their plasma lipid levels were in the normal range. The expression of apo E abolished the triglyceride-raising effect of apo CI.

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Cholesteryl Ester TransferProtein (CETP) Cholesteryl ester transfer protein (CETP) facilitates the exchange of neutral lipids among lipoproteins. Cholesteryl ester is transferred from HDL to VLDL and LDL. Mice normally lack plasma cholesteryl ester transfer activity, making them a suitable model for studying the function of CETP in vivo. The expression of human CETP in mice was found to cause a decrease in their HDL cholesterol levels (15). A surprising finding was, however, that LDL cholesterol did not increase in these transgenic mice, as was expected. The creation of mice expressing both human apo A-I and human CETP demonstratedthat human and mouse A-I proteins behave differently with CETP, with a more pronounced decrease in HDL cholesterol in the presence of human apo A-I (16). This implies that there could be some species-specific differences in apo A-I, with human A-I being a better substrate for CETP than mouse A-I.

Tissue-Specif ic Elements In addition to providing a new technique for the study of lipid metabolism, the work with transgenic mice has also been of great importance to the study of regulatory elements of the genes involved in lipid metabolism. In the human genome, there are two clusters of apolipoproteins. One is in chromosome 11, consisting of apo A-I, Clll and A-IV. Apo A-I and Clll are expressed both in the liver and in the intestine. The expression of apo A-IV is slightly different, since it is expressed mainly in the intestine with only a small fraction in the liver. The liverspecific expression of these genes seems to be controlled by different DNA regions, but the element responsible for intestinal expression seems to be shared by all of them (17, 18). This element is located in the intergenic region between apo Clll and A-IV. The other gene cluster is in chromosome 19, consisting of apo E, CI and CII. These apolipoproteins are normally expressed mainly in the liver. The element responsible for this liver-specific expression could be common for all three genes (14). The potential liver-specific element is located between apo CI and CII. The identification and isolation of different tissue-specific regions in the genome makes it possible to create transgenic animals that express a certain gene only in the designated tissue. In this way we can study the effect of the overexpression ofa gene in a specific tissue and also in a tissue where it is not normally expressed.

Conclusion With the advent of transgenic animal technology, we are able to study the effects of different genes in vivo in a controlled situation. Double positive mice can also be produced by mating different transgenic mouse lines with each other, and comparing lipoprotein metabolism in these mice either with mice expressing only one of the genes or with control mice. Using transgenic animal

technology, only overexpression of a gene can be studied, but with the advent of gene-targeting technology, the consequences of a decreased amount or a total lack of the expression of a gene can be studied (19). This new technology will increase our tools to better understand normal lipoprotein metabolism and also to determine more precisely the genetic defects underlying different primary dyslipidaemias.

References 1. Breslow JL. Apolipoprotein genetic variation and human disease. PhysiolRev 1988; 68: 85-1 32. 2. Jaenlsch R. Transgenic animals. Science 1988; 240: 1468-74. 3. Hofman SL, Russell DW, Brown MS, Goldstein JL, Hammer RE. Overexpression of low density lipoprotein (LDL) receptor eliminates LDL from plasma in transgenic mice. Science 1988; 239: 1277-81. 4. Goldstein JL, Brown MS. Familial hypercholesterolemia. In: Stanbury JB, Wyngaarden JB, fredrickson DS, Goldstein JL, Brown MS, eds. The metabolic basis of inherited disease. New York: McGraw-Hill, 1983: 672-712. 5. Yokode M, Hammer RE, lshlbashl S, Brown MS, Goldsteln JL. Diet-induced hypercholesterolemia in mice: prevention by overexpression of LDL receptors. Science 1990; 250: 1273-5. 6. Walsh A, Ito Y, Breslow JL. High levels of human apolipidoprotein A-I in transgenic mice result in increased plasma levels of small high density lipoprotein (HDL) particles comparable to human HDL,. J Biol Chem 1989; 264: 6488-94. 7. Rubin EM, lshlda BY, Cllft SM, Krauu RM. Expression of human apolipidoprotein A-I transgenic mice results in reduced plasma levels of murine apolipoprotein A-I and the appearance of two new high density lipoprotein size subclasses. Proc Natl Acad Sci USA 1991; 88: 434-8. 8. Chajek-Shaul T, Hayek T, Walsh A, Breslow JL. Expression of the human apolipoprotein A-I gene in transgenic mice alters high density lipoprotein (HDL) particles size distribution and diminished selective uptake of HDL cholesteryl esters. Proc NaN Acad Sci USA 1991; 88: 6731-5. 9. Rubln EM, Krauss RM, Spangler EA, Verstuyft JG, Cllft SM.Inhibition of early atherogenesis in transgenic mice by human apolipoprotein Al. Nature 1991; 353: 265-7. 10. Shlmano H, Katsukl M, Shimano M, et al. Overexpression of apolipoprotein E in transgenic mice: marked reduction in plasma lipoproteins except high density lipoprotein and resistance against diet induced hypercholesterolemia. Proc NatlAcad Sci USA 1992; 89: 1750-4. 11. Smith JD, Plump AS, Hayek T, Walsh A, Breslow JL. Accumulation of human apolipoprotein E in the plasma of transgenic mice. J Biol Chem 1990; 265: 14709-12. 12. It0 Y, Azrolan N, O’Connell A, Walsh A, Breslow JL. Hypertriglyceridemia as a result of human apo Clll gene expression in transgenic mice. Science 1990; 249: 790-3. 13. Aalto-SetaII K, Fisher EA, Chen X, et el. Mechanism for hypertriglyceridemia in human apo Clll transgenic mice: diminished VLDL fractional catabolic rate associated with increased apo Clll and reduced apo E on the particles. J Clin lnvest (in press). 14. Slmonett WS, Bucay N, Pltas RE, Lauer SJ,Taylor JM. Multiple tissue-specific elements control the apolipoprotein E/CI gene locus in transgenic mice. J Biol Chem 1991; 266: 8651-4. 15. Agelion LB, Walsh A, Hayek T, et al. Reduced high density lipoprotein cholesterol in human cholesteryl ester transfer

Transgenic Animals in Lipoprotein Research

the gene in the promoter of the adjacent convergently transcribed apo Clll gene. J Biol Chem (submitted). 18. Lauer SJ, Sirnonett WS, Bucay N, de Sllva HV, Taylor JM. Tissue-specific expression of the human apolipoprotein A-IV gene in transgenic mice. Circulation 1991; 84 Suppl: II18. 19. Capecchi M. The new mouse genetics altering the genome by gene targeting. Trends Gen 1989; 5: 70-6.

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protein transgenic mice. J Biol Chem 1991; 266: 10796-801. 16. Hayek T, Chajek-Shaul T, Walsh A, et al. An interaction between the human CETP and apo A-I genes in transgenic mice results in a profound CETP mediated depression of HDL cholesterol levels. J Clin invest 1992; 90: 505-1 0. 17. Walsh A, Azrolan N, Wang K, Marcigliano A, OConnell A, Breslow JL. Intestinal expression of the human apo A-I gene in transgenic mice is controlled by a DNA region 3' to

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Ann Med 24

Transgenic animals in lipoprotein research.

Lipid metabolism is a complex system involving the interaction of several lipid-transporting and -transferring serum proteins, enzymes regulating lipo...
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