Effect of increasing the expression of cholesterol transporters (StAR, MLN64, and SCP-2) on bile acid synthesis

Abstract

There are two major pathways of bile acid synthesis: the "neutral" pathway, initiated by highly regulated microsomal cholesterol 7 ␣ -hydroxylase (CYP7A1), and an "alternative" pathway, initiated by mitochondrial sterol 27-hydroxylase (CYP27A1). In hepatocyte cultures, overexpression of CYP7A1 increases bile acid synthesis by Ͼ 8-fold. However, overexpression of CYP27A1 in hepatocytes only increases it by 1.5-fold, suggesting that additional rate-limiting steps must be involved in the regulation of this pathway. The effects of intracellular cholesterol transport proteins on bile acid synthesis have been investigated in the current study. Under culture conditions in which the neutral pathway was inactive, selective overexpression of the gene encoding steroidogenic acute regulatory protein (StAR), MLN64 (StAR homolog protein), and sterol carrier protein-2 (SCP-2) led to 5.7-, 1.2-, and 1.7-fold increases, respectively, in the rates of bile acid synthesis in primary rat hepatocytes. Surprisingly, co-overexpression of MLN64 with StAR, SCP-2, or CYP7A1 blunted the upregulated bile acid synthesis by 48, 47, and 45%, respectively.These results suggest that MLN64, in its full-length form, is not responsible for the transport of cholesterol to the mitochondria or the endoplasmic reticulum, where CYP27A1 or CYP7A1 is located, respectively. -Ren, S., P. Hylemon, D. Marques, E. Hall, K. Redford, G. Gil, and W. M. Pandak. Effect of increasing the expression of cholesterol transporters (StAR, MLN64, and SCP-2) on bile acid synthesis.

Sulfated oxysterol, 25HC3S, is a potent regulator of lipid metabolism in human hepatocytes

“…The cells were sedimented by centrifugation and the pellets were washed three times by resuspension and sedimentation. Subcellular fractions, microsomal, cytosol, and nuclear, were isolated as previously described (19). The cellular or subcellular pellets were resuspended in 0.3 ml of PBS.…”

“…Microsomal fractions were isolated as previously described (19). Microsomal or total extracted proteins from the treated cells were separated on a 7.5% SDS-polyacrylamide denaturing gel.…”

“…When cells reached ~90% confluence, the oxysterols in DMSO or in ethanol (final concentration, 0.1%) and/or [1-14 C]acetate for cholesterol synthesis was added. Microsomal and cytosolic fractions were isolated from broken cells as described previously (19).…”

Overexpression of the cholesterol-binding protein MLN64 induces liver damage in the mouse

“…In the liver, MLN64 could mobilize cholesterol into mitochondria where it should be oxidized in the first step of the acidic pathway for bile acid synthesis [17] . Indeed, Pandak et al [18] and Ren et al [19,20] demonstrated that overexpression of the closest homologue of MLN64 in liver cells, StAR, leads to an important increase in bile acid synthesis both in vitro and in vivo. However, MLN64 overexpression in primary rat hepatocytes produced only a 20% increase in the rate of bile acid synthesis, suggesting that the hepatic expression of this protein is not a major determinant of the transport of cholesterol to the mitochondria during bile acid synthesis [19] .…”

The mammalian START domain protein family in lipid transport in health and disease

“…One of the first reported roles for STARD1 outside of steroidogenic cells was in cholesterol transfer across the mitochondrial membranes in the liver for initiation of bile acid synthesis by the alternative pathway. Overexpression of STARD1 significantly increases 27HC and bile acid synthesis in primary rat or mouse hepatocytes or human HepG2 hepatoma cells (Pandak et al 2002, Ren et al 2004a,b, Hall et al 2005. Enhanced rates of bile acid synthesis also occur in both rats and mice after overexpression of STARD1 in the liver, providing evidence for an in vivo function (Ren et al 2004a,b).…”