Lipoproteins

Lipids

(A) ACAT, a membrane integral enzyme is shown (yellow arrows) to convert free cholesterol present in the endoplasmic reticulum (ER) leaflets into cholesterol esters that remain within the membrane bilayer.

• ACAT1 is present in a variety of tissues, whereas
• ACAT2 is mainly expressed in enterocytes and hepatocytes.
• Both ACAT1 and ACAT2 are integral membrane proteins with multiple transmembrane domains and reside in the ER.
• They attach fatty acids from fatty acyl-CoA to the 3-hydroxyl group of membrane-associated cholesterol.

Newly synthesized cholesteryl esters are first partitioned into the ER membrane, where these enzymes reside, and are later transported for storage or secretion. In the liver and intestine, cholesteryl esters are transferred to apoB-lipoproteins that are being synthesized by MTP for secretion. Therefore, inhibition/deficiency of MTP is expected to increase cellular cholesteryl esters.

In contrast, Iqbal et al reported that Mttp gene deletion increases free cholesterol. They showed that MTP inhibition or genetic ablation had no effect on ACAT1 and ACAT2 mRNA and protein levels. Microsomal cholesteryl ester biosynthesis was, however, severely curtailed when MTP activity was reduced. Thus, MTP modulates cholesteryl ester synthesis by mechanisms other than transcriptional or translational control of enzymes critical for cholesterol ester biosynthesis.

To understand why MTP inhibition/deficiency leads to free cholesterol accumulation, they studied cholesterol ester biosynthesis in vitro using hepatic microsomes isolated from MTP deficient mice and observed significant reductions. Interestingly, supplementation of these microsomes with purified MTP restored cholesterol ester biosynthesis without affecting triglyceride and phospholipid biosynthesis. They also studied acute effects of MTP and ACAT inhibition using specific inhibitors in intestinal and hepatic cells. Individually, both inhibitors reduced cellular cholesterol esterification to similar extents and in combination they exhibited an additive effect. These studies indicated that MTP and ACAT affect different pathways in cholesterol ester biosynthesis.

To identify two different steps necessary for optimal cholesterol esterification, MTP and ACAT were expressed in cells that do not express these genes. Expression of ACAT increased cholesterol esterification; however, expression of MTP reduced cholesterol ester synthesis. Further studies showed that increased synthesis of cholesterol esters also requires apoB; expression of MTP and apoB48 in ACAT expressing cells increased cholesterol ester synthesis and secretion.

Therefore, cholesterol ester synthesis and lipoprotein biogenesis act in concert to maximize cholesterol ester biosynthesis. To delineate further how these pathways coordinate in optimal cholesterol ester synthesis and to identify molecular steps involved in this process, they reconstituted cholesterol ester synthesis in vitro using liver microsomes, purified MTP, and LDL. Enrichment of microsomes with cholesterol esters reduced cholesterol ester biosynthesis indicating that product accumulation inhibits this process. This inhibition was avoided when purified MTP and LDL was included in the reaction mixture. These studies suggest that MTP circumvents product inhibition by removing cholesterol esters and depositing them into apoB-lipoproteins.

Based on these studies, the role of MTP in cholesterol ester biosynthesis and consequences of MTP and/or ACAT deficiency on cellular cholesterol homeostasis can be explained as follows. Under normal conditions, ACAT synthesizes cholesteryl esters and MTP transfers both free and esterified cholesterol to apoB-lipoproteins. When MTP is limited, transfer of both free cholesterol and esterified cholesterol to apoB-lipoproteins is curtailed leading to accumulation of both esterified and free cholesterol. Accumulation of esterified cholesterol inhibits esterification by ACAT enzymes contributing to further accumulation of free cholesterol. Why doesn’t free cholesterol accumulate in the absence of ACAT? We speculate that in lipoprotein producing cells free cholesterol in the ER membrane is removed by the MTP thereby avoiding its accumulation.

• MTP is shown to transfer both free cholesterol and cholesteryl esters from the ER membranes to Apo B-lipoproteins in the ER lumen.
• It should be pointed out that MTP could transfer both free and esterified cholesterol to Apo B that is still associated with membranes.
• The thickness of orange arrows is meant to show that MTP most likely prefers cholesteryl esters over free cholesterol for transfer.

(B) In MTP deficient conditions, transfer of free and esterified cholesterol to Apo B-lipoproteins is reduced. Initially this might lead to accumulation of cholesteryl esters. When a high enough concentration of cholesteryl esters is achieved then ACAT activity is inhibited due to product inhibition leading to accumulation of free cholesterol.

(C) In the absence of ACAT activity, it is anticipated that cells accumulate more free cholesterol. Indeed, this is known to happen in cells that do not secrete Apo B-lipoproteins, such as macrophages. However, in cells that are able to synthesize Apo B-containing lipoproteins, MTP can transfer free cholesterol to lipoproteins avoiding excess free cholesterol accumulation in the ER membrane.

BMC, Nutrition & Metabolism

Review

February 2012

A physiological ratio of free cholesterol/phospholipids in the cellular membrane is necessary to maintain membrane fluidity and excess cellular free cholesterol is toxic to cells.

Hence, cellular free cholesterol levels are controlled by several pathways. One mechanism is to convert excess cellular free cholesterol and store it in ester forms. Two acyl-CoA:Cholesterol acyltransferase (ACAT) enzymes, ACAT1 and ACAT2, carry out similar cellular cholesterol esterification, but have different tissue distributions.

• ACAT1 is present in a variety of tissues, whereas
• ACAT2 is mainly expressed in enterocytes and hepatocytes.
• Both ACAT1 and ACAT2 are integral membrane proteins with multiple transmembrane domains and reside in the ER.
• They attach fatty acids from fatty acyl-CoA to the 3-hydroxyl group of membrane-associated cholesterol.

Newly synthesized cholesteryl esters are first partitioned into the ER membrane, where these enzymes reside, and are later transported for storage or secretion. In the liver and intestine, cholesteryl esters are transferred to apoB-lipoproteins that are being synthesized by MTP for secretion. Therefore, inhibition/deficiency of MTP is expected to increase cellular cholesteryl esters.

In contrast, Iqbal et al reported that Mttp gene deletion increases free cholesterol. They showed that MTP inhibition or genetic ablation had no effect on ACAT1 and ACAT2 mRNA and protein levels. Microsomal cholesteryl ester biosynthesis was, however, severely curtailed when MTP activity was reduced. Thus, MTP modulates cholesteryl ester synthesis by mechanisms other than transcriptional or translational control of enzymes critical for cholesterol ester biosynthesis.

To understand why MTP inhibition/deficiency leads to free cholesterol accumulation, they studied cholesterol ester biosynthesis in vitro using hepatic microsomes isolated from MTP deficient mice and observed significant reductions. Interestingly, supplementation of these microsomes with purified MTP restored cholesterol ester biosynthesis without affecting triglyceride and phospholipid biosynthesis. They also studied acute effects of MTP and ACAT inhibition using specific inhibitors in intestinal and hepatic cells. Individually, both inhibitors reduced cellular cholesterol esterification to similar extents and in combination they exhibited an additive effect. These studies indicated that MTP and ACAT affect different pathways in cholesterol ester biosynthesis.

To identify two different steps necessary for optimal cholesterol esterification, MTP and ACAT were expressed in cells that do not express these genes. Expression of ACAT increased cholesterol esterification; however, expression of MTP reduced cholesterol ester synthesis. Further studies showed that increased synthesis of cholesterol esters also requires apoB; expression of MTP and apoB48 in ACAT expressing cells increased cholesterol ester synthesis and secretion.

Therefore, cholesterol ester synthesis and lipoprotein biogenesis act in concert to maximize cholesterol ester biosynthesis. To delineate further how these pathways coordinate in optimal cholesterol ester synthesis and to identify molecular steps involved in this process, they reconstituted cholesterol ester synthesis in vitro using liver microsomes, purified MTP, and LDL. Enrichment of microsomes with cholesterol esters reduced cholesterol ester biosynthesis indicating that product accumulation inhibits this process. This inhibition was avoided when purified MTP and LDL was included in the reaction mixture. These studies suggest that MTP circumvents product inhibition by removing cholesterol esters and depositing them into apoB-lipoproteins.

Based on these studies, the role of MTP in cholesterol ester biosynthesis and consequences of MTP and/or ACAT deficiency on cellular cholesterol homeostasis can be explained as follows. Under normal conditions, ACAT synthesizes cholesteryl esters and MTP transfers both free and esterified cholesterol to apoB-lipoproteins. When MTP is limited, transfer of both free cholesterol and esterified cholesterol to apoB-lipoproteins is curtailed leading to accumulation of both esterified and free cholesterol. Accumulation of esterified cholesterol inhibits esterification by ACAT enzymes contributing to further accumulation of free cholesterol. Why doesn’t free cholesterol accumulate in the absence of ACAT? We speculate that in lipoprotein producing cells free cholesterol in the ER membrane is removed by the MTP thereby avoiding its accumulation.