Regulation of Intestinal Bile Acid Absorption in Health and Cholesterol-Related Disorders

The ileal apical sodium-dependent bile acid transporter (ASBT) is crucial for the enterohepatic
circulation of bile acids and plays a key role in maintaining bile acid, lipid, and cholesterol balance.
Emerging evidence suggests that ASBT is an attractive therapeutic approach to restore metabolic
functions and lower plasma cholesterol. This is particularly important in light of recent findings
indicating the need to lower cholesterol to very low levels in individuals with high risk for
developing cardiovascular diseases (CVDs) such as the veterans. Achieving such stringent target
remains challenging in many cases despite the use of cholesterol synthesis inhibitors and
blockers of cholesterol absorption. In this regard, dietary fatty acids are known to influence
metabolic functions as well as lipid and cholesterol homeostasis. A recent study showed that the
effectiveness of ASBT inhibition in restoring normal metabolic functions depends on the types of
fatty acids present in the diet. However, specific effects of different types of fatty acids on ASBT
function are not fully understood. Our recent novel data using resin-assisted capture (Acyl-RAC)
and click-chemistry based metabolic labeling approaches provided evidence that ASBT protein is
subject to s-acylation, a reversible post-translational modification where fatty acids attach to
cysteine residues of membrane proteins governing their membrane raft localization and function.
We have shown that incorporation of the unsaturated fatty acid oleate or omega-3 fatty acid
eicosapentaenoic acid (EPA) into ASBT was associated with a significant decrease in its function.
We have previously demonstrated that ASBT is regulated by post-transcriptional mechanisms
including its association with membrane lipid raft microdomains. Our preliminary findings showed
that feeding a diet rich with fish oil decreased ASBT function and association with lipid rafts in
mice. Thus, identifying mechanisms that target s-acylation could be beneficial in inhibiting ASBT
function as well as restoring normal lipid and cholesterol homeostasis. ASBT activity and
association with lipid rafts were increased in a transgenic mouse model (ISR2tg) with intestine-
specific overactivation of the Sterol Response Elements Binding Protein (SREBP2). Our recent
data demonstrated that ISR2tg mice develop hypercholesterolemia and severe hepatic
inflammation and fibrosis when fed a high fat high cholesterol diet and blocking bile acid
absorption by cholestyramine reduced the diet-induced liver injury. ASBT function and association
with lipid rafts are increased in ISR2tg mice. Based on these data, our proposed studies will test
the hypothesis that acylation is critical for ASBT function and association with lipid rafts and that
intestine-specific mechanisms are involved in regulating ASBT acylation (Specific aim 1). Studies
in Specific Aim 2 will elucidate mechanisms mediating the effects of omega-3 fatty acids on
ASBT function and s-acylation in in vitro models including enteroids and will examine their effects
on bile acid homeostasis as well as lipid and cholesterol metabolism in wildtype and ASBT
knockout mice. To investigate the beneficial effects of blocking ASBT acylation, our studies will
further utilize ISR2tg mice fed a high fat high cholesterol diet as a unique model to examine the
effects of feeding dietary omega-3 poly unsaturated fatty acids on ASBT acylation and metabolic
dysfunction (Specific Aim 2). Our findings represent a paradigm shift in our understanding of the
link between fatty acids and bile acid homeostasis. The proposed studies are highly significant
and are likely to unravel novel avenues pertaining to potential beneficial effects of blocking ASBT
function and acylation in restoring metabolic functions and cholesterol homeostasis.

Grant Number: 5I01BX000152-15
NIH Institute/Center: VA
Principal Investigator: Waddah Alrefai