**2. Species differences in lipoprotein metabolism**

#### **2.1. Species differences in lipoprotein profiles**

Fig. 1 shows lipoprotein profiles of mice, rabbits, and humans analysed with high performance liquid chromatography (HPLC). The main lipoprotein is LDL in human normal subjects but high-density lipoprotein (HDL) in wild-type mice. LDL is increased markedly in the plasma of patients with hypercholesterolemia (Yin, 2012) and WHHLMI rabbits, but lipoproteins that elute at the position of very low-density lipoprotein (VLDL) are increased in apoE-deficient mice, one of the most commonly used genetically modified mice (Piedrahita, 1992). Although serum lipid levels and the lipoprotein profile of apoE-KO mice vary depending on the colony, the lipoprotein profile of apoE-KO mice is similar to that of cholesterol-fed rabbits (Yin, 2012). In LDL receptor (LDLR)-deficient individuals, LDL increased markedly in patients (familial hypercholesterolemia) and rabbits (WHHLMI rabbits) despite a normal diet or chow, while plasma LDL levels were not so high in homozygous LDLR-KO mice fed standard chow as described by Ishibashi et al. (1993), although serum lipid levels and lipoprotein profiles of LDLR-KO mice vary in each colony similar to those of apoE-KO mice. After consumption of a cholesterolenriched diet, plasma cholesterol levels increased markedly in LDLR-KO mice, similar to FH patients and WHHL rabbits, but the increased lipoprotein fraction eluted at the position of VLDL (Ishibashi, 1994) and the lipoprotein profile was similar to that of cholesterol-fed rabbits (Yin, 2012). These differences in lipoprotein profiles between mice and humans or rabbits are probably due to the species differences in lipoprotein metabolism.

**Figure 1.** Lipoprotein profiles of a healthy human, rabbits, and mice. Lipoprotein profiles were analyzed with high performance liquid chromatography. Animals were fed standard chow. Red lines indicate cholesterol and blue lines indicate triglyceride

#### **2.2. Lipoprotein metabolism in mice and rats**

534 Lipoproteins – Role in Health and Diseases

lipoprotein metabolism.

metabolism.

**2. Species differences in lipoprotein metabolism** 

**2.1. Species differences in lipoprotein profiles** 

first knockout (KO) mice for lipoprotein metabolism were apolipoprotein (apo) E-KO mice, developed in 1992 (Zhang, 1992). Thereafter, numerous genetically modified mouse models were produced and these mice have contributed to a better understanding of lipoprotein metabolism. However, we should recognize that the lipoprotein metabolism of genetically modified mice is not entirely the same as that of humans, despite hyperlipidemia or hypercholesterolemia. In addition, recent studies have demonstrated different phenotypes manifested in mice and rabbits after the same gene transfer (Fan, 2003). The first transgenic rabbit was developed in 1985 (Hammer, 1985) and the first transgenic rabbit for lipoprotein metabolism, the hepatic lipase-overexpressing rabbit, was developed in 1994 (Fan, 1994). The differences in phenotype following gene transfer between mice and rabbits may be due to species differences in lipoprotein metabolism. When using animal models in experimental research, one has to be careful interpreting the results. Since other chapters explain in detail the functions of enzymes, apolipoproteins, and receptors relating to lipoprotein metabolism, this chapter concentrates on introducing various genetically modified animals and species differences in phenotype expression after gene modification for researchers wishing to study

Fig. 1 shows lipoprotein profiles of mice, rabbits, and humans analysed with high performance liquid chromatography (HPLC). The main lipoprotein is LDL in human normal subjects but high-density lipoprotein (HDL) in wild-type mice. LDL is increased markedly in the plasma of patients with hypercholesterolemia (Yin, 2012) and WHHLMI rabbits, but lipoproteins that elute at the position of very low-density lipoprotein (VLDL) are increased in apoE-deficient mice, one of the most commonly used genetically modified mice (Piedrahita, 1992). Although serum lipid levels and the lipoprotein profile of apoE-KO mice vary depending on the colony, the lipoprotein profile of apoE-KO mice is similar to that of cholesterol-fed rabbits (Yin, 2012). In LDL receptor (LDLR)-deficient individuals, LDL increased markedly in patients (familial hypercholesterolemia) and rabbits (WHHLMI rabbits) despite a normal diet or chow, while plasma LDL levels were not so high in homozygous LDLR-KO mice fed standard chow as described by Ishibashi et al. (1993), although serum lipid levels and lipoprotein profiles of LDLR-KO mice vary in each colony similar to those of apoE-KO mice. After consumption of a cholesterolenriched diet, plasma cholesterol levels increased markedly in LDLR-KO mice, similar to FH patients and WHHL rabbits, but the increased lipoprotein fraction eluted at the position of VLDL (Ishibashi, 1994) and the lipoprotein profile was similar to that of cholesterol-fed rabbits (Yin, 2012). These differences in lipoprotein profiles between mice and humans or rabbits are probably due to the species differences in lipoprotein

Fig. 2 shows a schematic diagram of lipoprotein metabolism in wild-type mice and rats. Dietary cholesterol is absorbed at the intestine mediated by Niemann-Pick C1-like 1 protein (NPC1L1) (Altmann, 2004) and ATP-binding cassette transporters (ABC) (Berge, 2000). Dietary fat is hydrolyzed to form monoglycerides and fatty acids in the intestine. These lipolytic products are translocated to the enterocyte membrane, and migrate to the endoplasmic reticulum. Overexpression of the tis7 gene in mice increases triglyceride absorption (Wang, 2009), and the transport of saturated fatty acids and cholesterol is decreased in fatty acid-binding protein (FABP)-KO mice (Newberry, 2009). Monoglycerides and fatty acids are re-esterified into triglycerides at the cytoplasmic surface of the endoplasmic reticulum. Synthesized triglycerides, cholesterol, and apolipoproteins are assembled in chylomicron particles. In the process of chylomicron assembly, MTP assists in binding lipids to apolipoproteins (Hussain, 2012). Since the apoB mRNA editing enzyme (apobec-1) functions at the intestinal wall, chylomicron particles contain apoB-48 as a major apolipoprotein but do not contain apoB-100. Chylomicron particles are released into the lacteal vessel. ApoB-48 is a marker of exogenous lipoprotein in humans and rabbits. At the capillary of peripheral tissue, chylomicron particles release free fatty acids (FFA) to adipose tissues mediated by lipoprotein lipase (LPL) and are converted to chylomicron remnants (Goldstein, 1983). Chylomicron remnants rapidly disappear from the circulation through apoE-receptors (apoER, remnant receptors) expressed in the liver. The metabolism of exogenous lipoproteins is preserved across species. In endogenous lipoprotein metabolism, cholesterol and other lipids synthesized in the liver are assembled in VLDL particles. Since apobec-1 is not expressed in the liver in humans and rabbits, VLDL particles contain apoB-100 but not apoB-48. However, VLDL particles of mice and rats contain both apoB-100 and apoB-48, because apobec-1 is expressed in the liver (Greeve, 1993). At the capillary in peripheral tissue, VLDL particles release FFA similar to chylomicron particles, which is then transformed into intermediatedensity lipoprotein (IDL) (Goldstein, 1983). Part of the remaining VLDL particles and/or partially catalyzed VLDL particles bind to VLDL receptors expressed in peripheral tissue (Takahashi, 2004). Finally, apoB-48-containing VLDL and IDL in mice and rats disappear from the circulation through apoER expressed in the liver similar to chylomicron remnants. Some IDL particles containing apoB-100 bind to LDLRs in the liver, the rest release fatty acids via hepatic lipase (HL) and are transformed into LDL. LDL particles bind to LDLRs expressed at the surface of somatic cells. Therefore, a marker of endogenous lipoproteins is apoB-100 in humans and rabbits but endogenous lipoproteins of mice and rats contain both apoB-100 and apoB-48. Since the fractional catabolic rate for apoB-48-containing lipoproteins is very high compared to that for apoB-100-containing lipoproteins (Li, 1996), concentrations of VLDL and LDL are very low in wild-type mice and rats compared to humans. In reverse cholesterol transport from peripheral tissue to the liver, high-density lipoproteins (HDLs) receive free cholesterol from macrophages through scavenger receptor type B-I (SR-BI) and ABCs such as ABCA1 (Rohrer, 2009). The free cholesterol transported from macrophages is esterified by lecithin:cholesterol acyltransferase (LCAT) in plasma. The esterified cholesterol in HDL particles is transferred to VLDL, IDL, and LDL in plasma by cholesterol ester transfer protein (CETP) in humans and rabbits (Son, 1986), while mice and rats do not have CETP activity in plasma (Agellon, 1991). Therefore, the cholesterol ester in HDL particles is not transferred to apoB-containing lipoproteins in mice and rats. This is one of the major reasons why HDL is the predominant lipoprotein in these two species. Circulating HDL particles bind to SR-BI in the liver. Therefore, the large difference in lipoprotein metabolism between mice / rats and humans / rabbits is characterized by both the expression of apobec-1 in the liver and absence of CETP in the plasma of mice and rats. Dyslipidemia develops when lipoprotein metabolism is impaired. For example, LDLR deficiency causes familial hypercholesterolemia, and impaired LPL function causes hypertriglyceridemia. A number of animal models including transgenic animals, knockout animals, and spontaneous mutant animals can develop hypercholesterolemia, hypertriglyceridemia, and postprandial hypertriglyceridemia. However, the lipoprotein profile of these mice is greatly different from that of human patients.

greatly different from that of human patients.

in humans and rabbits. At the capillary of peripheral tissue, chylomicron particles release free fatty acids (FFA) to adipose tissues mediated by lipoprotein lipase (LPL) and are converted to chylomicron remnants (Goldstein, 1983). Chylomicron remnants rapidly disappear from the circulation through apoE-receptors (apoER, remnant receptors) expressed in the liver. The metabolism of exogenous lipoproteins is preserved across species. In endogenous lipoprotein metabolism, cholesterol and other lipids synthesized in the liver are assembled in VLDL particles. Since apobec-1 is not expressed in the liver in humans and rabbits, VLDL particles contain apoB-100 but not apoB-48. However, VLDL particles of mice and rats contain both apoB-100 and apoB-48, because apobec-1 is expressed in the liver (Greeve, 1993). At the capillary in peripheral tissue, VLDL particles release FFA similar to chylomicron particles, which is then transformed into intermediatedensity lipoprotein (IDL) (Goldstein, 1983). Part of the remaining VLDL particles and/or partially catalyzed VLDL particles bind to VLDL receptors expressed in peripheral tissue (Takahashi, 2004). Finally, apoB-48-containing VLDL and IDL in mice and rats disappear from the circulation through apoER expressed in the liver similar to chylomicron remnants. Some IDL particles containing apoB-100 bind to LDLRs in the liver, the rest release fatty acids via hepatic lipase (HL) and are transformed into LDL. LDL particles bind to LDLRs expressed at the surface of somatic cells. Therefore, a marker of endogenous lipoproteins is apoB-100 in humans and rabbits but endogenous lipoproteins of mice and rats contain both apoB-100 and apoB-48. Since the fractional catabolic rate for apoB-48-containing lipoproteins is very high compared to that for apoB-100-containing lipoproteins (Li, 1996), concentrations of VLDL and LDL are very low in wild-type mice and rats compared to humans. In reverse cholesterol transport from peripheral tissue to the liver, high-density lipoproteins (HDLs) receive free cholesterol from macrophages through scavenger receptor type B-I (SR-BI) and ABCs such as ABCA1 (Rohrer, 2009). The free cholesterol transported from macrophages is esterified by lecithin:cholesterol acyltransferase (LCAT) in plasma. The esterified cholesterol in HDL particles is transferred to VLDL, IDL, and LDL in plasma by cholesterol ester transfer protein (CETP) in humans and rabbits (Son, 1986), while mice and rats do not have CETP activity in plasma (Agellon, 1991). Therefore, the cholesterol ester in HDL particles is not transferred to apoB-containing lipoproteins in mice and rats. This is one of the major reasons why HDL is the predominant lipoprotein in these two species. Circulating HDL particles bind to SR-BI in the liver. Therefore, the large difference in lipoprotein metabolism between mice / rats and humans / rabbits is characterized by both the expression of apobec-1 in the liver and absence of CETP in the plasma of mice and rats. Dyslipidemia develops when lipoprotein metabolism is impaired. For example, LDLR deficiency causes familial hypercholesterolemia, and impaired LPL function causes hypertriglyceridemia. A number of animal models including transgenic animals, knockout animals, and spontaneous mutant animals can develop hypercholesterolemia, hypertriglyceridemia, and postprandial hypertriglyceridemia. However, the lipoprotein profile of these mice is

Genetically Modified Animal Models for Lipoprotein Research 537

**Figure 2.** Lipoprotein metabolism in mice and rats (wild-type). Abbreviations: ACAT, acyl-CoA:cholesterol acyltransferase; apoB, apolipoprotein B; CETP, cholesterol ester transfer protein; Chyl, chylomicron; CR, chylomicron remnant; DGAT2, acyl-Co-A:diacylglycerol acyltransferase 2; ER, apoE receptor; FABP, fatty acids-binding protein; FFA, free fatty acids; H, high-density lipoprotein; HL, hepatic lipase; I48, intermediate-density lipoprotein (IDL) with apoB-48; I100, IDL with apoB-100; L, low-density lipoprotein (LDL); LCAT, lecithin:cholesterol acyltransferase; LPL, lipoprotein lipase; LR, LDL receptor; M, macrophage; MTP, microsomal triglyceride transfer protein; NPC1L1, Niemann-Pick C1-like 1; PLTP, phospholipid transfer protein; SR-BI, scavenger receptor type B-I; V48, very lowdensity lipoprotein (VLDL) with apoB-48; V100, VLDL with apoB-100; VR, VLDL receptor
