Biochemistry In complex diseases that are systemic throughout the body, there is
ID: 210774 • Letter: B
Question
Biochemistry
In complex diseases that are systemic throughout the body, there is often no way to model the disease process without the use of in vivo animal model systems. The study of lipid metabolism and atherosclerosis in the body relies on animal research to test hypotheses and develop new treatments to help improve human health.
You are working in a research team using several mouse strains that are gene knockouts for key proteins in cholesterol and apolipoprotein metabolism. Consider the following mouse strains, which have had genes turned off, referred to as “knockouts”:
- Apolipoprotein E knockout mice (Apoe/) that cannot make ApoE protein
- LDL receptor -/- mice( Ldlr/ ) that do not make LDL receptor.
- Apob100/100 mice expressing only ApoB-100 and not ApoB-48
- Apob48/48 mice expressing only ApoB-48 and not ApoB-100.
- Apob+/+ normal mice that express both forms of ApoB.
The two hypercholesterolemic mouse models, the apo E–deficient mouse (Apoe/) and the LDL receptor–deficient mouse (Ldlr/) have been used to study atherogenesis, or the development of fatty plaques at the start of atherosclerosis. On a normal mouse chow diet, Apoe/ mice have the highest cholesterol levels, with total cholesterol levels of ~400 mg/dL. Chow-fed Ldlr/ mice have mildly increased plasma cholesterol levels (175 to 225 mg/dL) from an accumulation of LDL and develop only minimal atherosclerotic lesions. Apo B-48 is the predominant apolipoprotein in the VLDL remnants of Apoe/ mice, whereas apo B-100 predominates in the LDL of Ldlr/ mice. The following table summarizes the characteristics observed in these two hypercholesterolemic mouse models.
These two knockout strains of mice have allowed scientists to investigate the genetic influence on atherosclerosis. Moreover, these mice can be fed controlled diets with different fat profiles and their health status can be closely monitored, which cannot be done in a human population. However, there are drawbacks to these models. In the case of the Ldlr-/- mice, there is a minimal development of the atherosclerotic plaques, even after 9-12 months, which is very late in a mouse life span. Conversely, Apoe-/- mice have incredibly high plasma cholesterol levels, far higher than normally observed in people, and it is mostly ApoB-48 associated cholesterol. Humans with high cholesterol normally have high ApoB-100, which is different from these mice. To understand the implications, mice expressing only ApoB-100 or ApoB-48 were also developed. These two strains have similar total and HDL cholesterol to Apob+/+ normal mice, but the Apob48/48 mice have significantly lower plasma TAG, while the Apob100/100 have significantly higher plasma TAG.
Based on your knowledge of Apolipoproteins in the different types of cholesterol, explain why you think there are these differences in lipoprotein and apolipoprotein composition between the four mouse strains of Apoe-/-, Ldlr-/-, Apob48/48, and Apob100/100 mice:
1. How would the differences in ApoB protein expression between human and the Ldlr-/- mice be a potential problem to using this mouse model to describe human disease?
2. Does the composition of the Apob48/48 and Apob100/100 cholesterol make sense based on what we know about ApoB-48 and ApoB-100?
Apoe Mice 400 mg/dL, 5 times above controls Greatly increased Modestly increased Decreased 3 months streaks, 8 months plaques Many large plaques after 14 weeks Ldir Mice 200 mg/dL, 2-3 times higher than controls Modestly increased Greatly increased Modestly increased Phenotype Hypercholesterolemia (cholesterol in the blood) VLDL IDL/LDLe HDL Spontaneous fatty plaques Medium plaques after 12 weeks on high cholesterol diet High fat diet induced plaquesExplanation / Answer
Lipoproteins are complexes of lipid emulsified by a diverse set of amphipathic proteins called apolipoprotein. In humans and certain genetically modified mice, circulating levels of cholesterol in low density lipoprotein (LDL) are directly related to development of atherosclerosis and subsequent cardiovascular disease (CVD) while levels in high density lipoprotein (HDL) are inversely related. Due to specific sequence and structural features, apolipoproteins mediates on-particle processing of the lipid as well as its targeted delivery throughout the body. Therefore, the “lipoproteome” of a given particle plays a major role in determining its protective or pathological fate.
PART 1:
The LDL receptor plays a critical role in the regulation of plasma LDL levels, and the Loss of LDL receptor function leads to decreased LDL catabolism and elevated LDL levels. LDL receptor levels are affected by diet, hormones, and most dramatically, by mutations in the LDL receptor locu .
The post-translational fate of apoB, the major protein component, is explained by multiple mechanisms. In human and rat hepatoma cell lines, a large proportion of newly synthesized apoB is degraded within the secretory pathway. Thus, the rate of apoB secretion, and hence, VLDL secretion, from the liver is determined by the proportion of apoB that escapes co- or post-translational degradation In addition, reuptake of newly secreted lipoproteins has also been proposed to regulate the net output of APoB.
The presence or absence of a functional LDL receptor affects the production of lipoprotein, if the cross between the apoB isolated from wild-type mice and mice lacking a functional LDL receptor (Ldlr–/–).Ldlr–/– mice shows a decrease in LDL clearance and a marked increase in plasma APoB levels. This indicates that the LDL receptor is involved in determining the post-translational fate of APoB by increasing presecretory APoB degradation and mediating reuptake of nascent lipoprotein particles.
PART -2:
Apolipoprotein (apo) B is an essential component of VLDL, LDL, and chylomicrons. ApoB exists in 2 forms, apoB100 and apoB48 and both are the products of the same gene. ApoB100 comprises 4536 amino acids and synthesized in the liver and secreted into the circulation as a structural component of VLDL. ApoB48 is 48% of full length APoB and is formed as a result of posttranslational editing of ApoB mRNA by the APoB editing complex (APoBEC), which changes Gln at codon to stop codon.
ApoB48 is synthesized in the small intestine and is used for the packaging of lipids into chylomicrons. Whereas human liver makes exclusively apoB100, a large proportion of message in the mouse liver is edited and consequently mice produce both apoB48 and apoB100 from the liver. In addition to maintaining the structural integrity of lipoprotein particles, apoB100 also functions as a ligand for the LDLR and is therefore a primary determinant of circulating LDL cholesterol levels expected that the mice producing apoB100 would model defective apolipoprotein B100 in humans by accumulating binding-defective LDL in plasma. However, it has been researched that the APoB100/ 100- mice y lower than normal, total plasma cholesterol and HDL cholesterol, and the amount of plasma LDL was not different from in wild-type mice.
Other point is that these 2 regions are not essential for APoB100 binding to the LDLR in vivo. The interpretation shows that, as it is complicated because mice normally have very little APoB100-containing LDL particles in circulation. In addition to this, the production of APoB48 from the liver and the efficient clearance of apoB48-containing remnants mediated by apoE make the metabolism of apoB100 difficult to study in vivo in mice. The study explains that the composition of APoB 48/48 and APoB 100/100 cholesterol makes the sense.
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